Interferon-associated antigen binding proteins and uses thereof

ABSTRACT

The present invention relates to novel interferon-associated antigen binding proteins as well as nucleic acids, vectors and vector systems encoding such interferon-associated antigen binding proteins. The present invention also relates to compositions comprising such interferon-associated antigen binding proteins, nucleic acids, vectors and vector systems. The novel interferon-associated antigen binding proteins afford beneficial improvements over the current state of the art, for example in that they effectively disrupt viral replication and thereby reduce HBV viral load. Thus, the present invention also provides medical uses of such interferon-associated antigen binding proteins, nucleic acids, vectors, vector systems and compositions, e.g., in the treatment of hepatitis B virus (HBV) infection and/or for decreasing one or more symptoms of HBV infection in a subject. The present invention further provides host cells comprising such nucleic acids, vectors and vector systems as well as methods of making the interferon- associated antigen binding proteins according to the invention using said host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application ofPCT/EP2020/083745 filed 27 Nov. 2020, which claims priority to EuropeanPatent Application No. EP19306552.1 filed 3 Dec. 2019 and EuropeanPatent Application No. EP19306573.7 filed 4 Dec. 2019, the entiredisclosures of which are herein incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on 3 Jun. 2022, isnamed DFMP-134-PCT-US_ST25.txt and is 187 Kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to novel interferon-associated antigenbinding proteins based on the agonistic antiCD40 antibody CP870,893 aswell as nucleic acids, vectors and vector systems encoding suchinterferon-associated antigen binding proteins. The present inventionalso relates to compositions comprising such interferon-associatedantigen binding proteins, nucleic acids, vectors and vector systems. Thenovel interferon-associated antigen binding proteins afford beneficialimprovements over the current state of the art, for example in that theyeffectively disrupt viral replication and thereby reduce HBV viral load.Thus, the present invention also provides medical uses of suchinterferon-associated antigen binding proteins, nucleic acids, vectors,vector systems and compositions, e.g., in the treatment of hepatitis Bvirus (HBV) infection and/or for decreasing one or more symptoms of HBVinfection in a subject. The present invention further provides hostcells comprising such nucleic acids, vectors and vector systems as wellas methods of making the interferon-associated antigen binding proteinsaccording to the invention using said host cells.

BACKGROUND

HBV infects more than 300 million people worldwide and is a common causeof liver disease and liver cancer (Liang (2009) Hepatology 49:S13). HBVis a small DNA virus with unusual features similar to retroviruses,which replicates through an RNA intermediate (pre-genomic RNA, pgRNA)and can integrate into the host genome. The unique features of the HBVreplication cycle confer a distinct ability of the virus to persist ininfected cells. HBV infection leads to a wide spectrum of liver diseaseranging from acute (including fulminant hepatic failure) to chronichepatitis, cirrhosis and hepatocellular carcinoma. Acute HBV infectioncan be either asymptomatic or present with symptomatic acute hepatitis.90-95% of children and 5-10% of adults infected with HBV are unable toclear the virus and become chronically infected. Many chronicallyinfected persons have mild liver disease with little or no long-termmorbidity or mortality. Other individuals with chronic HBV infectiondevelop active disease, which can progress to cirrhosis and livercancer. These patients require careful monitoring and warranttherapeutic intervention.

Novel methods for treating HBV infection by modulating HBV infection ina cell are needed. In particular, methods for effectively disruptingviral replication, reducing HBV viral load of HBV-infected cells,reducing transcription of covalently closed circular HBV DNA inHBV-infected cells, and/or reducing the amount of pre-genomic HBV RNA inHBV-infected cells are needed.

SUMMARY OF THE INVENTION

The invention relates to an interferon-associated antigen bindingprotein comprising (I) an agonistic anti-CD40 antibody or an agonisticantigen binding fragment thereof, and (II) an Interferon (IFN) or afunctional fragment thereof, wherein the agonistic anti-CD40 antibody,or the agonistic antigen binding fragment thereof, comprises

-   (a) three light chain complementarity determining regions (CDRs)    that are at least 90% identical to the CDRL1, CDRL2 and CDRL3    sequences within SEQ ID NO 3; and three heavy chain CDRs that are at    least 90% identical to the CDRH1, CDRH2 and CDRH3 sequences within    SEQ ID NO 6; wherein each CDR is defined in accordance with the    Kabat definition, the Chothia definition, the AbM definition, or the    contact definition of CDR; preferably wherein each CDR is defined in    accordance with the CDR definition of Kabat or the CDR definition of    Chothia;-   (b) three light chain complementarity determining regions (CDRs)    that are identical to the CDRL1, CDRL2 and CDRL3 sequences within    SEQ ID NO 3; and three heavy chain CDRs that are identical to the    CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6; wherein each    CDR is defined in accordance with the Kabat definition, the Chothia    definition, the AbM definition, or the contact definition of CDR;    preferably wherein each CDR is defined in accordance with the CDR    definition of Kabat or the CDR definition of Chothia;-   (c) a heavy chain or a fragment thereof comprising a complementarity    determining region (CDR) CDRH1 that is at least 90% identical to SEQ    ID NO 56, a CDRH2 that is at least 90% identical to SEQ ID NO 57,    and a CDRH3 that is at least 90% identical to SEQ ID NO 58; and    -   a light chain or a fragment thereof comprising a CDRL1 that is        at least 90% identical to SEQ ID NO 52, a CDRL2 that is at least        90% identical to SEQ ID NO 53, and a CDRL3 that is at least 90%        identical to SEQ ID NO 54;-   (d) a heavy chain or a fragment thereof comprising a complementarity    determining region (CDR) CDRH1 that is identical to SEQ ID NO 56, a    CDRH2 that is identical to SEQ ID NO 57, and a CDRH3 that is    identical to SEQ ID NO 58; and a light chain or a fragment thereof    comprising a CDRL1 that is identical to SEQ ID NO 52, a CDRL2 that    is identical to SEQ ID NO 53, and a CDRL3 that is identical to SEQ    ID NO 54;-   (e) a light chain variable region V_(L) comprising the sequence as    set forth in SEQ ID NO 51, or a sequence at least 90% identical    thereto; and/or a heavy chain variable region V_(H) comprising the    sequence as set forth in SEQ ID NO 55, or a sequence at least 90%    identical thereto;-   (f) a Fab region heavy chain comprising an amino acid sequence as    set forth in SEQ ID NO 12, or a sequence at least 90% identical    thereto; or-   (g) a light chain (LC) that comprises a sequence as set forth in SEQ    ID NO 3, or a sequence at least 90% identical thereto; and/or a    heavy chain (HC) that comprises a sequence selected from the group    consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49 and SEQ ID NO    48, or a sequence at least 90% identical thereto.

According to this aspect of the invention, the IFN or the functionalfragment thereof is selected from the group consisting of a Type I IFN,a Type II IFN and a Type III IFN, or a functional fragment thereof. In apreferred embodiment, the Type I IFN, or the functional fragmentthereof, is IFNα or IFNβ, or a functional fragment thereof.

According to one embodiment, the IFN or the functional fragment thereofis IFNα2a, or a functional fragment thereof. Preferably, the IFNα2acomprises the sequence as set forth in SEQ ID NO 17, or a sequence atleast 90% identical thereto.

According to another embodiment, the IFN or the functional fragmentthereof is IFNβ, or a functional fragment thereof. In a preferredembodiment, the IFNβ comprises the sequence as set forth in SEQ ID NO14, or a sequence at least 90% identical thereto.

According to a further embodiment, the IFN or the functional fragmentthereof is fused to a light chain of the agonistic anti-CD40 antibody orthe agonistic antigen binding fragment thereof. Preferably, the IFN orthe functional fragment thereof is fused to a C-terminus of a lightchain of the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof.

According to another embodiment, the IFN or the functional fragmentthereof is fused to a heavy chain of the agonistic anti-CD40 antibody orthe agonistic antigen binding fragment thereof. In a preferredembodiment, the IFN or the functional fragment thereof is fused to aC-terminus of a heavy chain of the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof.

According to another embodiment, the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof, and the IFN or thefunctional fragment thereof, are fused to each other via a linker. In apreferred embodiment, the linker comprises a sequence as set forth inSEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.

According to another embodiment, the interferon-associated antigenbinding protein comprises a sequence selected from the group consistingof SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32,SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37,SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42,SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46 and SEQ ID NO 47.

According to further embodiment, the interferon-associated antigenbinding protein is an interferon-fused agonistic anti-CD40 antibody oran interferon-fused agonistic antigen binding fragment thereofcomprising one of the sequence combinations disclosed in Table 8.

According to another aspect, the invention relates to a nucleic acidencoding the interferon-associated antigen binding protein according tothe invention. In a preferred embodiment, the nucleic acid furtherencodes a secretory signal peptide.

According to a further aspect, the invention relates to a vectorcomprising said nucleic acid.

According to another aspect, the invention relates to a vector systemcomprising

-   (I) a first vector comprising a nucleic acid encoding the IFN, or    the functional fragment thereof, fused to a light chain of the    agonistic anti-CD40 antibody, or the agonistic antigen binding    fragment thereof, of the interferon-associated antigen binding    protein of the present invention; and a second vector comprising a    nucleic acid encoding a heavy chain of the agonistic anti-CD40    antibody, or the agonistic antigen binding fragment thereof, of the    interferon-associated antigen binding protein of the present    invention; or-   (II) a first vector comprising a nucleic acid encoding the IFN, or    the functional fragment thereof, fused to a heavy chain of the    agonistic anti-CD40 antibody, or the agonistic antigen binding    fragment thereof, of the interferon-associated antigen binding    protein of the present invention; and a second vector comprising a    nucleic acid encoding a light chain of the agonistic anti-CD40    antibody, or the agonistic antigen binding fragment thereof, of the    interferon-associated antigen binding protein of the present    invention.

According to another aspect, the invention relates to a composition,preferably a pharmaceutical composition, comprising aninterferon-associated antigen binding protein, a nucleic acid, a vector,or a vector system according to the invention.

According to further aspect, the invention relates to a host cellcomprising a nucleic acid, a vector, or a vector system according to theinvention. According to another aspect, the invention relates to amethod of making an interferon-associated antigen binding proteinaccording to the invention, comprising culturing said host cell andrecovering said interferon-associated antigen binding protein.

According to another aspect, the invention relates to aninterferon-associated antigen binding protein, a nucleic acid, a vector,a vector system, or a composition according to the invention for use asa medicament.

According to yet another aspect, the invention relates to aninterferon-associated antigen binding protein, a nucleic acid, a vector,a vector system, or a composition according to the invention for use intreating hepatitis B virus (HBV) infection and/or for decreasing one ormore symptoms of HBV infection in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : This schematic drawing depicts exemplary interferon-associatedantigen binding protein formats. The interferon-associated antigenbinding protein is an interferon-fused agonistic anti-CD40 antibody oran interferon-fused agonistic antigen binding fragment thereof. IFNs areassociated via linkers to different positions on the antibody or theantigen binding fragment thereof: N-terminal or C-terminal part of thelight chain (LC) or the heavy chain (HC). In particular, IFNs are chosenfrom Type I, Type II and Type III interferon families.

FIG. 2A depicts an exemplary map of a pcDNA3.1 plasmid encoding SEQ IDNO 32 under the control of the pCMV promoter. The nucleic acid sequenceencoding for SEQ ID NO 32 (SEQ ID NO 59) is also shown on the right.Italic: signal peptide sequence; black color: CP870,893 heavy chaincoding sequence; underlined: HL linker coding sequence; bold: IFNβcoding sequence.

FIG. 2B shows examples of SDS PAGE in reduced conditions of some IFAs,with IFNα or IFNβ fused either at the heavy chain or the light chain.Migration of the parental CP870,893 is also shown on the left.

FIGS. 3A-3B graphically depict a dose dependent effect of a number ofIFA molecules with IFNβ fusions on activating the CD40-mediated NFκBpathway reporter assay in HEK-Blue™ CD40L cells. FIG. 3A shows examplesof anti-CD40 activities for IFAs with IFNβ fused to the C-terminal partof the heavy chain (HC). FIG. 3B shows examples of anti-CD40 activitiesfor IFAs with IFNβ fused to the N-terminal part of the LC (IFA34) or theHC (IFA36) and the corresponding fusions on the C-terminal part (IFA35and IFA37). Purification yield of the latter group of IFAs was very low,thus to test their activity, the supernatants from HEK transfected cellswere used and serially diluted to evaluate the anti-CD40 activity onHEK-Blue™ CD40L cells.

FIGS. 3C-3D graphically depict a dose dependent effect of a number ofIFA molecules with IFNβ fusions on activating the Type I IFN- pathway inreporter HEK-Blue-IFN-α/β cells. FIG. 3C shows examples of IFN activityfor IFAs with IFNβ fused to the C-terminal part of the HC. FIG. 3D showsexamples of IFN activity for IFAs with IFNβ fused to the N-terminal partof the LC (IFA34) or the HC (IFA36) and the corresponding fusions on theC-terminal part (IFA35 and IFA37). The same supernatants from HEKtransfected cells as in FIG. 3B were used and serially diluted toevaluate the IFN activity. Parental antibody CP870,893 was used asnegative control and recombinant human IFNβ was used as positivecontrol. NS: Non Stimulated.

FIG. 4A graphically depicts a dose effect of a number of IFA moleculeswith IFNα fusions on activating the CD40-mediated NFκB pathway reporterassay in HEK-Blue™ CD40L cells.

FIG. 4B graphically depicts a dose effect of a number of IFA moleculeswith IFNα fusions on activating the Type I IFN-mediated pathway inreporter HEK-Blue-IFN-α/β cells. The activity of Pegasys is indicated inthe insert in the lower right corner.

FIG. 4C graphically depicts the effect of IFA molecules with IFNαfusions and HL linker on HC (IFA38) or LC (IFA39) on activating theCD40-mediated NFκB pathway reporter assay in HEK-Blue™ CD40L cells.

FIG. 4D graphically depicts the effect of IFA38 and IFA39 on activationof the Type I IFN-pathway in reporter HEK-Blue-IFNα/β cells.

FIG. 5 depicts the effect of IFNβ based IFAs in a dose dependent manneron HBeAg release from primary hepatocytes infected with HBV. IFA1,IFA12: fusion of IFNβ to the C-terminus of LC via HL or RL linkers,respectively. IFA2 and IFA13: fusion of IFNβ_C17S to the C-terminus ofthe LC via HL or RL linkers, respectively.

FIG. 6A depicts the effect of IFA25, IFA26 and IFA27 in a dose dependentmanner on HBeAg release from primary human hepatocytes infected by HBV.

FIG. 6B depicts the effect of IFA28, IFA29 and IFA30 in a dose dependentmanner on HBeAg release from primary human hepatocytes infected by HBV.

FIG. 6C depicts a dose response anti-viral activity (HBeAg release) ofIFAs with HL linker (IFA38 and IFA39) on HBV-infected PHHs.

FIGS. 6D-6H depict a dose response anti-viral activity of 4 IFAmolecules with fusion to IFNα via a peptide linker on primary humanhepatocytes infected with HBV. FIG. 6D: Cartoon illustrating the studydesign. FIG. 6E: Effect of IFAs on HBeAg release in comparison toPegasys. FIG. 6F: Effect of IFAs on HBsAg release in comparison toPegasys. FIG. 6G: Effect of IFAs on pgRNA levels in comparison toPegasys. FIG. 6H: Effect of IFAs on CXCL10 release in comparison toPegasys.

FIG. 7 depicts results from an in vitro Cytokines Release Assay of HumanWhole Blood Cells (WBCs): Example of data obtained after stimulation ofWBCs from 4 healthy volunteer donors. WBC were left Non-Stimulated (NS),treated with LPS (10 ng/mL) or with IFA1 (1 µg/mL) for 24 h.Supernatants were collected and submitted to cytokines releasequantification using the MSD u-Plex kit for human cytokines. Resultsrepresent the mean of two independent stimulations from each donor. Theprofile of CXCL10 (IP10), IL6, IL1β and TNFα are shown.

Tables 9a-b: These tables summarize data obtained after in vitrostimulation of whole blood cells (WBCs) obtained from healthyvolunteers. Each IFA was tested on WBCs from four different donors. WBCswere left Non-Treated (NT), treated with LPS (10 ng/mL) or with IFAs (1µg/mL) for 24 h. Supernatants were collected and submitted to cytokinesrelease quantification using the MSD u-Plex kit for human cytokines.Results represent the mean of two independent stimulations from eachdonor and are expressed in pg/mL (nd: not detected).

FIG. 8 : Pharmacokinetic profile of IFA25, IFA26, IFA27, IFA28, IFA29,and IFA30 after 0.5 mg/kg (IFAs) or 0.3 mg/kg (Pegasys) intravenousbolus injection to mice. Data expressed as mean +/- SD onsemi-logarithmic scale. Samples were collected up to 10 days afteradministration. ELISA assay using anti-IFNα as secondary antibody forquantification method was used for IFA27, IFA29 and IFA30 (FIG. 8A) andfor IFA25, IFA26 and IFA28 (FIG. 8B). ELISA assay using anti-IgG2 assecondary antibody for quantification method was used for IFA25 andIFA27 (FIG. 8C). FIG. 8D: Pegasys quantification was done using humanIFNα matched antibody pairs. The marked line (LLOQ) denotes the limit ofdetection for the Pegasys assay.

Table 10A: PK Report Summary: PK parameters for CP870,893, IFA27, IFA29and IFA30 following single intravenous administration of 0.5 mg/kg tomale CD1 Swiss mice. PK parameters for CP870,893 were explored in a7-day experiment and those for IFA27, IFA29 and IFA30 in 10-dayexperiments (quantification for IFA27 was performed using 2 differentELISA approaches).

Table 10B: PK parameters for CP870,893, Pegasys and for three differentIFAs (IFA25, IFA26 and IFA28) following single intravenous bolusadministration of 0.5 mg/kg to male CD1 Swiss mice. PK parameters forCP870,893 and IFA25, IFA26, IFA28 and Pegasys were explored in 21-dayexperiments (quantification for IFA25 was performed using 2 differentELISA approaches).

FIG. 9A depicts CD40 agonistic activity in a dose dependent manner ofIFA50 and IFA51 with no Fc region in comparison to the parentalanti-CD40 antibody in reporter HEK-Blue™ CD40L cells. FIG. 9B depictsthe IFNα activity in a dose dependent manner of IFA50 and IFA51 inreporter HEK-Blue™ hIFN-α/β cells. FIG. 9C: Effect of IFA50 and IFA51 onHBeAg release from HBV-infected PHHs.

FIG. 10A depicts CD40 agonistic activity in a dose dependent manner ofIFNs based IFA49, in comparison to parental anti-CD40 antibody, inHEK-Blue™ CD40L reporter cells. IFA49 corresponds to fusion of IFNε tothe HC via a peptide linker. FIG. 10B depicts the IFN activity in a dosedependent manner of IFA49 on reporter HEK-Blue™ hIFN-α/β reporter cellswhich are activated by Type I interferons. FIG. 10C: Effect of IFA49 onHbeAg release from HBV-infected PHHs.

FIG. 11A depicts CD40 agonistic activity in a dose dependent manner ofIFNω based IFA46, in comparison to parental anti-CD40 antibody, inHEK-Blue™ CD40L reporter cells. IFA46 corresponds to fusion of IFNω tothe LC via a peptide linker. FIG. 11B depicts the IFN activity in a dosedependent manner of IFA46 on reporter HEK-Blue™ hIFN-α/β reporter cellswhich are activated by Type I interferons. FIG. 11C: Effect of IFA46 onHbeAg release from HBV-infected PHHs.

FIG. 12A depicts CD40 agonistic activity in a dose dependent manner ofIFNγ based IFAs (IFA42 and IFA43), in comparison to parental anti-CD40antibody, in HEK-Blue™ CD40L reporter cells. IFA42 corresponds to fusionof IFNγ to the LC via a peptide linker and IFA43 correspond to fusion ofIFNγ to the HC via a peptide linker. FIG. 12B depicts the IFN activityin a dose dependent manner of IFA42 and IFA43 in reporter HEK-Blue-hIFNγcells. FIG. 12C: Effect of IFA42 and IFA43 on HbeAg release fromHBV-infected PHHs.

FIG. 13A depicts CD40 agonistic activity in a dose dependent manner ofIFNλ, based IFAs (IFA44 and IFA45), in comparison to parental anti-CD40antibody, in HEK-Blue™ CD40L reporter cells. IFA44 corresponds to fusionof IFNλ to the LC via a peptide linker and IFA45 correspond to fusion ofIFNλ to the HC via a peptide linker. FIG. 13B depicts the IFN activityin a dose dependent manner of IFA44 and IFA45 in reporterHEK-Blue-hIFNλ, cells. FIG. 13C: Effect of the IFNλ based IFAs (IFA44and IFA45) on HbeAg release from HBV-infected PHHs.

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

DETAILED DESCRIPTION

The present invention is based in part on the discovery of a therapythat is based on the use of “interferon-associated antigen-bindingproteins”, variants or derivatives thereof comprising (I) an agonisticanti-CD40 antibody or an agonistic antigen binding fragment thereof, and(II) an interferon (IFN) or a functional fragment thereof in hepatitis Bvirus (HBV) therapy. Said interferon-associated antigen-binding proteinsinhibit transcription of hepatitis B virus covalently closed circularDNA (cccDNA) into pre-genomic HBV RNA (pgRNA) in HBV-infected cells,inhibit release of hepatitis B e-antigen (HBeAg) from HBV-infectedcells, and enhance the IFN pathway in uninfected and HBV infectedhepatocytes, in particular in uninfected and HBV infected primary humanhepatocytes and in a synergistic fashion. HBV therapy comprisingadministering an interferon-associated antigen-binding protein to anHBV-infected cell, or a subject infected with HBV, is provided.

The invention may be more readily understood in the light of theselected terms defined below.

As used herein, the term “CD40” refers to “Cluster of differentiation40”, a member of the tumor necrosis factor receptor (TNFR) superfamily.CD40 is a costimulatory protein found on antigen presenting cells (e.g.,B cells, dendritic cells, monocytes), hematopoietic precursors,endothelial cells, smooth muscle cells, epithelial cells, as well as themajority of human tumors (Grewal & Flavell, Ann. Rev. Immunol., 1996,16: 111-35; Toes & Schoenberger, Seminars in Immunology, 1998, 10(6):443-8). The binding of the natural ligand CD154 (CD40L) on T_(H) cellsto CD40 activates antigen presenting cells and induces a variety ofdownstream effects. The TNF-receptor associated factor adaptor proteinsTRAF1, TRAF2, TRAF6 and TRAF5 interact with CD40 and serve as mediatorsof the signal transduction. Ultimately, CD40 signaling activates boththe canonical and the noncanonical NF-κB pathways.

Agonistic anti-CD40 Antibodies and Antigen Binding Fragments Thereof

As used herein, the term “antibody” refers to immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated VH or V_(H)) and a heavy chain constant region (CHor C_(H)). The heavy chain constant region comprises three domains, CH1,CH2 and CH3. Each light chain comprises a light chain variable region(abbreviated VL or V_(L)) and a light chain constant region (CL orC_(L)). The light chain constant region comprises one domain (CL1). TheVH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions (CDRs)”,interspersed with regions that are more conserved, termed “frameworkregions” (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Framework regions can aid inmaintaining the proper conformation of the CDRs to promote bindingbetween the antigen binding region and an antigen.

The most commonly used immunoglobulin for therapeutic applications isimmunoglobulin G (or IgG), a tetrameric glycoprotein. In anaturally-occurring immunoglobulin, each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one light (about25 kDa) and one heavy chain (about 50-70 kDa). The amino-terminalportion of each chain includes a variable region of about 100 to 110 ormore amino acids primarily responsible for antigen recognition. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Immunoglobulins can beassigned to different classes depending on the amino acid sequence ofthe constant domain of their heavy chains.

Heavy chains are classified as mu (µ), delta (δ), gamma (γ), alpha (α),and epsilon (ε), and define the antibody’s isotype as IgM, IgD, IgG,IgA, and IgE, respectively. Several of these may be further divided intosubclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Different isotypes have different effector functions; for example, IgG1and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC)activity. In preferred embodiments, the agonistic antiCD40 antibodies oragonistic antigen binding fragments thereof comprised in theinterferon-associated antigen binding proteins according to theinvention are of the IgG class. In more preferred embodiments, theagonistic antiCD40 antibodies or agonistic antigen binding fragmentsthereof comprised in the interferon-associated antigen binding proteinsaccording to the invention are of the IgG1 or IgG3 subclasses. Inspecifically preferred embodiments, the agonistic antiCD40 antibodies oragonistic antigen binding fragments thereof comprised in theinterferon-associated antigen binding proteins according to theinvention are of the IgG1 subclass. In other more preferred embodiments,the agonistic antiCD40 antibodies or agonistic antigen binding fragmentsthereof comprised in the interferon-associated antigen binding proteinsaccording to the invention are of the IgG2 or IgG4 subclasses. Inspecifically preferred embodiments, the agonistic antiCD40 antibodies oragonistic antigen binding fragments thereof comprised in theinterferon-associated antigen binding proteins according to theinvention are of the IgG2 subclass.

Human light chains are classified as kappa (κ) and lambda (λ) lightchains. Accordingly, in some embodiments, the agonistic antiCD40antibodies or agonistic antigen binding fragments thereof comprised inthe interferon-associated antigen binding proteins according to theinvention comprise a light chain of the κ class. In other embodiments,the agonistic antiCD40 antibodies or agonistic antigen binding fragmentsthereof comprised in the interferon-associated antigen binding proteinsaccording to the invention comprise a light chain of the λ class. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, wherein the heavy chainadditionally includes a “D” region of about 10 more amino acids. Seegenerally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)).

The term “antibody” further includes, but is not limited to, monoclonalantibodies, bispecific antibodies, minibodies, domain antibodies,synthetic antibodies (sometimes referred to as “antibody mimetics”),chimeric antibodies, humanized antibodies, human antibodies, andfragments thereof, respectively. Unless otherwise indicated, the term“antibody” includes, in addition to antibodies comprising twofull-length heavy chains and two full-length light chains, derivatives,variants, antigen binding fragments, and muteins thereof, examples ofwhich are described below.

As used herein, the term “agonistic CD40 antibody” or “agonisticanti-CD40 antibody” refers to an antibody that binds to CD40 andmediates CD40 signaling. In a preferred embodiment, it binds to humanCD40. As described below, binding to CD40 may be determined usingsurface plasmon resonance, preferably using the BIAcore® system. Theagonistic anti-CD40 antibody may increase one or more CD40 activities byat least about 20% when added to a cell, tissue or organism expressingCD40. In some embodiments, the antibody activates CD40 activity by atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, or atleast 85%. CD40 activity of the agonistic anti-CD40 antibody may bemeasured using a whole blood surface molecule upregulation assay orusing an in vitro reporter cell assay, e.g., using HEK-Blue™ CD40L cells(InvivoGen Cat. #: hkb-cd40), as described in greater detail in ExampleI. These reporter cells were generated by stable transfection of HEK293cells with the human CD40 gene and an NFκB-inducible secreted embryonicalkaline phosphatase (SEAP) construct to measure the activity of CD40agonists. Stimulation of CD40 leads to NFκB activation and thus toproduction of SEAP, which can be detected in the supernatant usingchromogenic substrates such as QUANTI-Blue™.

In the context of the present invention, the interferon-associatedantigen binding proteins activate both the CD40 and an IFN pathway. Incertain embodiments, the interferon-associated antigen binding proteinactivates the CD40 pathway with an EC₅₀ of less than 400, 300, 200, 150,100, 70, 60, 50, 40, 30, 25, 20, or 15 ng/mL. In more specificembodiments, the interferon-associated antigen binding protein activatesthe CD40 pathway with an EC₅₀ ranging from 10 to 200 ng/mL. In even morespecific embodiments, the interferon-associated antigen binding proteinactivates the CD40 pathway with an EC₅₀ ranging from 10 to 50 ng/mL,preferably 10 to 30 ng/mL.

Exemplary light and heavy chain sequences of the agonistic anti-CD40antibody CP870,893 are shown in Table 7.

As used herein, the term “agonistic antigen binding fragment” of anagonistic anti-CD40 antibody refers to a fragment of an agonisticanti-CD40 antibody that retains one or more functional activities of theoriginal antibody, such as the ability to bind to and act as an agonistof CD40 signaling in a cell, e.g., it mediates CD40 pathway signaling.Such fragment may compete with the intact antibody for binding to CD40.

Agonistic antigen binding fragments of an agonistic anti-CD40 antibodycan be produced by recombinant DNA techniques, or can be produced byenzymatic or chemical cleavage of an anti-CD40 antibody. Agonisticantigen binding fragments include, but are not limited to, a Fabfragment, a diabody (heavy chain variable domain on the same polypeptideas a light chain variable domain, connected via a short peptide linkerthat is too short to permit pairing between the two domains on the samechain), a Fab′ fragment, a F(ab′)₂ fragment, a Fv fragment, domainantibodies and single-chain antibodies, and can be derived from anymammalian source, including but not limited to human, mouse, rat,camelid or rabbit.

The term “variable region” or “variable domain” refers to a portion ofthe light and/or heavy chains of an antibody, typically includingapproximately the amino-terminal 120 to 130 amino acids in the heavychain and about 100 to 110 amino terminal amino acids in the lightchain. Variable regions of different antibodies differ extensively inamino acid sequence even among antibodies derived from the same speciesor of the same class. Exemplary V_(L) and V_(H) domain sequences of theagonistic anti-CD40 antibody CP870,893 are shown in Table 1. Thevariable region of an antibody typically determines specificity of aparticular antibody for its target as it contains the CDRs. Table 1 alsoshows exemplary CDR sequences of the agonistic anti-CD40 antibodyCP870,893.

TABLE 1 Anti-CD40 antibody heavy/light chain variable regions and CDRsof the agonistic anti-CD40 antibody CP870,893. Bold italic sequencescorrespond to CDR regions according to the Kabat definition Anti-CD40antibody regions Sequence antiCD40 Antibody V_(L) domain (SEQ ID NO 51)DIQMTQSPSSVSASVGDRVTITC RASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANIFPLTFGGGTKVEIK antiCD40 Antibody CDRL1 (SEQ ID NO 52) RASQGIYSWLA antiCD40Antibody CDRL2 (SEQ ID NO 53) TASTLQS antiCD40 Antibody CDRL3 (SEQ ID NO54) QQANIFPLT antiCD40 Antibody V_(H) domain (SEQ ID NO 55)QVQLVQSGAEVKKPGASVKVSCKASGYTF TGYYMH WVRQAPGQGLEWMGWINPDSGGTNY AQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYC ARDQPLGYCTNGVCSYFDY WGQGTLVTVSS antiCD40Antibody CDRH1 (SEQ ID NO 56) TGYYMH antiCD40 Antibody CDRH2 (SEQ ID NO57) WINPDSGGTNYAQKFQG antiCD40 Antibody CDRH3 (SEQ ID NO 58)DQPLGYCTNGVCSYFDY

Delineation of a CDR and identification of residues comprising thebinding site of an antibody may be accomplished by solving the structureof the antibody and/or solving the structure of the antibody-ligandcomplex. This can be accomplished by any of a variety of techniquesknown to those skilled in the art, such as X-ray crystallography.Various methods of analysis can be employed to identify or approximatethe CDR regions. Examples of such methods include, but are not limitedto, the Kabat definition, the Chothia definition, the AbM definition andthe contact definition.

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. See, e.g.,Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothiadefinition is similar to the Kabat definition, but the Chothiadefinition takes into account positions of certain structural loopregions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986);Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses anintegrated suite of computer programs produced by Oxford Molecular Groupthat model antibody structure. See, e.g., Martin et al., Proc Natl AcadSci (USA), 86:9268-9272 (1989); “AbM™, A Computer Program for ModelingVariable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. TheAbM definition models the tertiary structure of an antibody from primarysequence using a combination of knowledge databases and ab initiomethods, such as those described by Samudrala et al., “Ab Initio ProteinStructure Prediction Using a Combined Hierarchical Approach,” inPROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). Thecontact definition is based on an analysis of the available complexcrystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45(1996).

In certain embodiments, the complementarity determining regions (CDRs)of the light and heavy chain variable regions of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof, can begrafted to framework regions (FRs) from the same, or another, species.In certain embodiments, the CDRs of the light and heavy chain variableregions of an agonistic anti-CD40 antibody, or an agonistic antigenbinding fragment thereof, can be grafted to consensus human FRs. Tocreate consensus human FRs, in certain embodiments, FRs from severalhuman heavy chain or light chain amino acid sequences are aligned toidentify a consensus amino acid sequence. In certain embodiments, theFRs of the heavy chain or light chain of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof, are replacedwith the FRs from a different heavy chain or light chain. In certainembodiments, rare amino acids in the FRs of the heavy and light chainsof an agonistic anti-CD40 antibody, or an agonistic antigen bindingfragment thereof, are not replaced, while the rest of the FR amino acidsare replaced. Rare amino acids are specific amino acids that are inpositions in which they are not usually found in FRs. In certainembodiments, the grafted variable regions from an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof, can be usedwith a constant region that is different from the constant region of anagonistic anti-CD40 antibody, or an agonistic antigen binding fragmentthereof. In certain embodiments, the grafted variable regions are partof a single chain Fv antibody. CDR grafting is described, e.g., in U.S.Pat. Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761,5,565,332, 5,585,089, and 5,530,101, and in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyenet al., Science, 239:1534-1536 (1988), Winter, FEBS Letts., 430:92-94(1998), which are hereby incorporated by reference for any purpose.

An “Fc” region typically comprises two heavy chain fragments comprisingthe C_(H)2 and C_(H)3 domains of an antibody. The two heavy chainfragments are held together by two or more disulfide bonds and byhydrophobic interactions of the C_(H)3 domains.

A “Fab fragment” comprises one full-length light chain as well as theC_(H)1 and variable regions of one heavy chain (the combination of theV_(H) and C_(H)1 regions is referred to herein as “fab region heavychain”).

A “Fab′ fragment” comprises one light chain and a portion of one heavychain that contains the VH domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 andNo. 5,260,203, the disclosures of which are incorporated by reference.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody cantarget the same or different antigens.

An antibody or antigen binding protein, such as an interferon-associatedantigen binding protein according to the invention, preferably binds toits target antigen with a dissociation constant (K_(d)) of ≤10⁻⁷ M. Theantibody or antigen binding protein binds its antigen with “highaffinity” when the K_(d) is ≤5 × 10⁻⁹ M, and with “very high affinity”when the K_(d) is ≤5 × 10⁻¹⁰ M. More preferably, the antibody or antigenbinding protein has a K_(d) of ≤10⁻⁹ M. In some embodiment, the off-rateis <1 × 10⁻⁵. In other embodiments, the antibody or antigen bindingprotein will bind to human CD40 with a K_(d) of between about 10⁻⁹ M and10⁻¹³ M, and in yet another embodiment the antibody or antigen bindingprotein will bind with a K_(d) ≤5 × 10⁻¹⁰. As will be appreciated by oneof skill in the art, in some embodiments, any or all of the antigenbinding fragments can bind to CD40. Preferably, said constants aredetermined using surface plasmon resonance, more preferably using theBIAcore® system.

The term “surface plasmon resonance” means an optical phenomenon thatallows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore® system (BIAcore International AB,a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson et al. (1993) Ann. Biol. Clin.51:19-26. The term “K_(on)” means the on rate constant for associationof a binding protein (e.g., an antibody or antigen binding protein) tothe antigen to form the, e.g., antigen binding protein/antigen complex.The term “K_(on)”, or “on-rate” also means “association rate constant”,or “ka”, as is used interchangeably herein. This value indicating thebinding rate of a binding protein to its target antigen or the rate ofcomplex formation between a binding protein, e.g., an antibody or anantigen binding protein, and antigen also is shown by the equationbelow:

The term “K_(off)”, or “off-rate”, means the off rate constant fordissociation, or “dissociation rate constant”, of a binding protein(e.g., an antibody or antigen binding protein) from the, e.g., antigenbinding protein/antigen complex as is known in the art. This valueindicates the dissociation rate of a binding protein, e.g., an antibodyor an antigen binding protein, from its target antigen or separation ofAb-Ag complex over time into free antibody and antigen as shown by theequation below:

The terms “K_(d)” and “equilibrium dissociation constant” means thevalue obtained in a titration measurement at equilibrium, or by dividingthe dissociation rate constant (K_(off)) by the association rateconstant (K_(on)). The association rate constant, the dissociation rateconstant and the equilibrium dissociation constant, are used torepresent the binding affinity of a binding protein (e.g., an antibodyor an antigen binding protein) to an antigen. Methods for determiningassociation and dissociation rate constants are well known in the art.Using fluorescence-based techniques offers high sensitivity and theability to examine samples in physiological buffers at equilibrium.Other experimental approaches and instruments such as a BIAcore®(biomolecular interaction analysis) assay, can be used (e.g., instrumentavailable from BIAcore International AB, a GE Healthcare company,Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay)assay, available from Sapidyne Instruments (Boise, Id.), can also beused.

An antigen binding protein according to the invention may bind to onetarget with an affinity at least one order of magnitude, preferably atleast two orders of magnitude higher than for a second target.

The term “target” refers to a molecule or a portion of a moleculecapable of being bound by an antigen binding protein. In certainembodiments, a target can have one or more epitopes. It will thereforebe understood that the target may serve as “antigen” for the “antigenbinding protein” of the present invention.

The term “epitope” includes any determinant capable of being bound by anantigen binding protein, such as an antibody. An epitope is a region ofan antigen that is bound by an antigen binding protein that targets thatantigen, and when the antigen is a protein, includes specific aminoacids that directly contact the antigen binding protein. Most often,epitopes reside on proteins, but in some instances can reside on otherkinds of molecules, such as nucleic acids. Epitope determinants caninclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl or sulfonyl groups, and can havespecific three-dimensional structural characteristics, and/or specificcharge characteristics. Generally, antibodies specific for a particulartarget antigen will preferentially/specifically recognize an epitope onthe target antigen in a complex mixture of proteins and/ormacromolecules.

In exemplary embodiments, the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof forming part (I) of theinterferon-associated antigen binding proteins of the inventioncomprises three light chain complementarity determining regions (CDRs)that are at least 90% identical to the CDRL1, CDRL2 and CDRL3 sequenceswithin SEQ ID NO 3; and three heavy chain CDRs that are at least 90%identical to the CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.The agonistic anti-CD40 antibody, or the agonistic antigen bindingfragment thereof, may also comprise three light chain complementaritydetermining regions (CDRs) that are identical to the CDRL1, CDRL2 andCDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs that areidentical to the CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6. Insuch embodiments, each CDR is defined in accordance with the Kabatdefinition, the Chothia definition, the AbM definition, or the contactdefinition of CDR; preferably wherein each CDR is defined in accordancewith the CDR definition of Kabat or the CDR definition of Chothia. Inparticular embodiments, each CDR is defined in accordance with the Kabatdefinition. In other particular embodiments, each CDR is defined inaccordance with the Chothia definition.

Alternatively, the agonistic anti-CD40 antibody, or the agonisticantigen binding fragment thereof forming part (I) of theinterferon-associated antigen binding proteins of the inventioncomprises (a) a heavy chain or a fragment thereof comprising acomplementarity determining region (CDR) CDRH1 that is at least 90%, atleast 95%, at least 98% or at least 99% identical to SEQ ID NO 56, aCDRH2 that is at least 90%, at least 95%, at least 98% or at least 99%identical to SEQ ID NO 57, and a CDRH3 that is at least 90%, at least95%, at least 98% or at least 99% identical to SEQ ID NO 58; and (b) alight chain or a fragment thereof comprising a CDRL1 that is at least90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO52, a CDRL2 that is at least 90%, at least 95%, at least 98% or at least99% identical to SEQ ID NO 53, and a CDRL3 that is at least 90%, atleast 95%, at least 98% or at least 99% identical to SEQ ID NO 54.

In some embodiments, the agonistic anti-CD40 antibody, or the agonisticantigen binding fragment thereof, comprises (a) a heavy chain or afragment thereof comprising a complementarity determining region (CDR)CDRH1 that is identical to SEQ ID NO 56, a CDRH2 that is identical toSEQ ID NO 57, and a CDRH3 that is identical to SEQ ID NO 58; and (b) alight chain or a fragment thereof comprising a CDRL1 that is identicalto SEQ ID NO 52, a CDRL2 that is identical to SEQ ID NO 53, and a CDRL3that is identical to SEQ ID NO 54.

More specifically the agonistic anti-CD40 antibody, or the agonisticantigen binding fragment thereof, comprises a light chain variableregion V_(L) comprising the sequence as set forth in SEQ ID NO 51, or asequence at least 90%, at least 95%, at least 98% or at least 99%identical thereto; and/or a heavy chain variable region V_(H) comprisingthe sequence as set forth in SEQ ID NO 55, or a sequence at least 90%,at least 95%, at least 98% or at least 99% identical thereto.

The interferon-associated antigen binding proteins of the invention mayalso comprise an agonistic anti-CD40 antibody or an agonistic antigenbinding fragment thereof, comprising a Fab region heavy chain comprisingan amino acid sequence as set forth in SEQ ID NO 12, or a sequence atleast 90%, at least 95%, at least 98% or at least 99% identical thereto.

In some embodiments, the agonistic anti-CD40 antibody or the agonisticantigen binding fragment thereof comprises a light chain (LC) thatcomprises a sequence as set forth in SEQ ID NO 3, or a sequence at least90%, at least 95%, at least 98% or at least 99% identical thereto;and/or a heavy chain (HC) that comprises a sequence selected from thegroup consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49, SEQ ID NO 12and SEQ ID NO 50, or a sequence at least 90%, at least 95%, at least 98%or at least 99% identical thereto.

In more specific embodiments, the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof comprises a light chain (LC)that comprises a sequence as set forth in SEQ ID NO 3, or a sequence atleast 90%, at least 95%, at least 98% or at least 99% identical thereto;and/or a heavy chain (HC) that comprises a sequence as set forth in SEQID NO 6, or a sequence at least 90%, at least 95%, at least 98% or atleast 99% identical thereto.

In more specific embodiments, the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof comprises a light chain (LC)that comprises a sequence as set forth in SEQ ID NO 3, or a sequence atleast 90%, at least 95%, at least 98% or at least 99% identical thereto;and/or a heavy chain (HC) that comprises a sequence as set forth in SEQID NO 9, or a sequence at least 90%, at least 95%, at least 98% or atleast 99% identical thereto.

In other more specific embodiments, the agonistic anti-CD40 antibody orthe agonistic antigen binding fragment thereof comprises a light chain(LC) that comprises a sequence as set forth in SEQ ID NO 3, or asequence at least 90%, at least 95%, at least 98% or at least 99%identical thereto; and/or a heavy chain (HC) that comprises a sequenceas set forth in SEQ ID NO 49, or a sequence at least 90%, at least 95%,at least 98% or at least 99% identical thereto.

In other more specific embodiments, the agonistic anti-CD40 antibody orthe agonistic antigen binding fragment thereof comprises a light chain(LC) that comprises a sequence as set forth in SEQ ID NO 3, or asequence at least 90%, at least 95%, at least 98% or at least 99%identical thereto; and/or a heavy chain (HC) that comprises a sequenceas set forth in SEQ ID NO 12, or a sequence at least 90%, at least 95%,at least 98% or at least 99% identical thereto.

In other more specific embodiments, the agonistic anti-CD40 antibody orthe agonistic antigen binding fragment thereof comprises a light chain(LC) that comprises a sequence as set forth in SEQ ID NO 3, or asequence at least 90%, at least 95%, at least 98% or at least 99%identical thereto; and/or a heavy chain (HC) that comprises a sequenceas set forth in SEQ ID NO 50, or a sequence at least 90%, at least 95%,at least 98% or at least 99% identical thereto.

Variants and Derivatives of Interferon-Associated Antigen BindingProtein or Components Thereof

A “variant” of a polypeptide (e.g., an interferon-associated antigenbinding protein, an interferon-fused agonistic anti-CD40 antibody or aninterferon-fused agonistic antigen binding fragment thereof, anantibody, an antigen binding protein, or an IFN, or components thereof)comprises an amino acid sequence wherein one, two, three, four, five ormore amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. Preferably, the variant comprises up to ten insertions,deletions and/or substitutions, more preferably up to eight insertions,deletions and/or substitutions. More specifically, the variant maycomprise up to ten, more preferably up to eight insertions. The variantmay also comprise up to ten, more preferably up to eight deletions. Ineven more preferred embodiments, the variant comprises up to tensubstitutions, most preferably up to eight substitutions. In someembodiments, these substitutions are conservative amino acidsubstitution as described below.

A “variant” of a polynucleotide sequence (e.g., RNA or DNA) comprisesone or more mutations within the polynucleotide sequence relative toanother polynucleotide sequence, wherein one, two, three, four, five ormore nucleic acid residues are inserted into, deleted from and/orsubstituted into the nucleic acid sequence. Preferably, the variantcomprises up to ten insertions, deletions and/or substitutions, morepreferably up to eight insertions, deletions and/or substitutions. Morespecifically, the variant may comprise up to ten, more preferably up toeight insertions. The variant may also comprise up to ten, morepreferably up to eight deletions. In even more preferred embodiments,the variant comprises up to ten substitutions, most preferably up toeight substitutions. Said one, two, three, four, five or more mutationscan cause one, two, three, four, five or more amino acid exchangeswithin the amino acid sequence the variant encodes for as compared toanother amino acid sequence (i.e. a “non-silent mutation”). Variantsalso include nucleic acid sequences wherein one, two, three, four, fiveor more codons have been replaced by their synonyms which does not causean amino acid exchange and is thus called a “silent mutation”.

The term “identity” or “homology”, in the context of variants ofpolypeptide or nucleotide sequences, refers to a relationship betweenthe sequences of two or more polypeptide molecules or two or morenucleic acid molecules, as determined by aligning and comparing thesequences. “Percent identity” means the percent of identical residuesbetween the amino acids or nucleotides in the compared molecules and iscalculated based on the size of the smallest of the molecules beingcompared. Preferably, identity is determined over the full length of asequence. It is understood that the expression “at least 80% identical”,includes embodiments wherein the claimed sequence is at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identical to thereference sequence. The expression “at least 90 % identical” includesembodiments wherein the claimed sequence is at least 90%, at least 91 %,at least 92 %, at least 93%, at least 94%, at least 95 %, at least 96 %,at least 97 %, at least 98% or at least 99% identical to the referencesequence.

For the calculation of percent identity, gaps in alignments (if any) arepreferably addressed by a particular mathematical model or computerprogram (i.e., an “algorithm”). Methods that can be used to calculatethe identity of the aligned nucleic acids or polypeptides include thosedescribed in Computational Molecular Biology, (Lesk, A. M., ed.), 1988,New York: Oxford University Press; Biocomputing Informatics and GenomeProjects, (Smith, D. W., ed.), 1993, New York: Academic Press; ComputerAnalysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G.,eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, SequenceAnalysis in Molecular Biology, New York: Academic Press; SequenceAnalysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York:M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math.48:1073.

In calculating percent identity, the sequences being compared aretypically aligned in a way that gives the largest match between thesequences. One example of a computer program that can be used todetermine percent identity is the GCG program package, which includesGAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics ComputerGroup, University of Wisconsin, Madison, WI). The computer algorithm GAPis used to align the two polypeptides or polynucleotides for which thepercent sequence identity is to be determined. The sequences are alignedfor optimal matching of their respective amino acid or nucleotide (the“matched span”, as determined by the algorithm). A gap opening penalty(which is calculated as 3× the average diagonal, wherein the “averagediagonal” is the average of the diagonal of the comparison matrix beingused; the “diagonal” is the score or number assigned to each perfectamino acid match by the particular comparison matrix) and a gapextension penalty (which is usually ⅒ times the gap opening penalty), aswell as a comparison matrix such as PAM 250 or BLOSum 62 are used inconjunction with the algorithm. In certain embodiments, a standardcomparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequenceand Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff etal., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSum62 comparison matrix) is also used by the algorithm.

Examples of parameters that can be employed in determining percentidentity for polypeptides or nucleotide sequences using the GAP programare the following:

-   Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453-   Comparison matrix: BLOSum 62 from Henikoff et al., 1992, supra-   Gap Penalty: 12 (but with no penalty for end gaps)-   Gap Length Penalty: 4-   Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (GAP program) canbe adjusted if so desired to result in an alignment that spans at least50 or at least 100, preferably the entire length, of contiguous aminoacids of the target polypeptide.

Conservative amino acid substitutions can encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues can be divided into classes based on commonside chain properties:

-   1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;-   2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;-   3) acidic: Asp, Glu;-   4) basic: His, Lys, Arg;-   5) residues that influence chain orientation: Gly, Pro; and-   6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions can involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues can be introduced, for example, into regions of ahuman antibody that are homologous with non-human antibodies, or intothe non-homologous regions of the molecule.

In making changes to the interferon-associated antigen binding protein,according to certain embodiments, the hydropathic index of amino acidscan be considered. Each amino acid has been assigned a hydropathic indexon the basis of its hydrophobicity and charge characteristics. They are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine(-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine(-4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids can be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. In certainembodiments, the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith its immunogenicity and antigenicity, i.e., with a biologicalproperty of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1);glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);phenylalanine (-2.5) and tryptophan (-3.4). In making changes based uponsimilar hydrophilicity values, in certain embodiments, the substitutionof amino acids whose hydrophilicity values are within ±2 is included, incertain embodiments, those which are within ±1 are included, and incertain embodiments, those within ±0.5 are included.

Exemplary amino acid substitutions are set forth in Table 2.

TABLE 2 Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile LysArg, 1,4 Diamino-butyric Acid, Gln, Asn Arg Met Leu, Phe, Ile Leu PheLeu, Val, Ile, Ala, Tyr Leu Pro Ala, Gly Ala Ser Thr, Ala, Cys Thr ThrSer Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu,Phe, Ala, Norleucine Leu

In light of the present invention, a skilled artisan will be able todetermine suitable variants of the interferon-associated antigen bindingproteins as set forth herein using well-known techniques. In certainembodiments, one skilled in the art can identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In certain embodiments,even areas that can be important for biological activity or forstructure can be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues which are important for activity or structure insimilar proteins. One skilled in the art can opt for chemically similaramino acid substitutions for such predicted important amino acidresidues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarproteins or protein domains. In view of such information, one skilled inthe art can predict the alignment of amino acid residues ofinterferon-associated antigen binding protein, an antibody or an antigenbinding fragment thereof or an interferon or a functional fragmentthereof as described herein with respect to its three dimensionalstructure. In certain embodiments, one skilled in the art can choose notto make radical changes to amino acid residues predicted to be on thesurface of the protein, since such residues can be involved in importantinteractions with other molecules. Moreover, one skilled in the art cangenerate test variants containing a single amino acid substitution ateach desired amino acid residue. The variants can then be screened usingactivity assays known to those skilled in the art. Such variants can beused to gather information about suitable variants. For example, if onediscovered that a change to a particular amino acid residue resulted indestroyed, undesirably reduced, or unsuitable activity, variants withsuch a change can be avoided. In other words, based on informationgathered from such experiments, one skilled in the art can readilydetermine the amino acids where further substitutions should be avoidedeither alone or in combination with other mutations.

According to certain embodiments, amino acid substitutions are thosewhich: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (5) confer ormodify other physicochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) can be made in the naturally-occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts). In certain embodiments, aconservative amino acid substitution typically may not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Branden& J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al., Nature, 354:105 (1991), which are each incorporatedherein by reference.

The term “derivative” refers to a molecule that includes a chemicalmodification other than an insertion, deletion, or substitution of aminoacids (or nucleic acids). In certain embodiments, derivatives comprisecovalent modifications, including, but not limited to, chemical bondingwith polymers, lipids, or other organic or inorganic moieties. Incertain embodiments, a chemically modified interferon-associated antigenbinding protein can have a greater circulating half-life than aninterferon-associated antigen binding protein that is not chemicallymodified. In certain embodiments, a chemically modifiedinterferon-associated antigen binding protein can have improvedtargeting capacity for desired cells, tissues, and/or organs. In someembodiments, a derivative interferon-associated antigen binding proteinis covalently modified to include one or more water-soluble polymerattachments, including, but not limited to, polyethylene glycol,polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Pat.Nos: 4,640,835, 4,496,689, 4,301,144, 4,670,417,4,791,192 and 4,179,337.In certain embodiments, a derivative interferon-associated antigenbinding protein comprises one or more polymer, including, but notlimited to, monomethoxy-polyethylene glycol, dextran, cellulose, orother carbohydrate based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of suchpolymers.

In certain embodiments, a derivative of an interferon-associated antigenbinding protein as described herein is covalently modified withpolyethylene glycol (PEG) subunits. In certain embodiments, one or morewater-soluble polymer is bonded at one or more specific position, forexample at the amino terminus, of a derivative. In certain embodiments,one or more water-soluble polymer is randomly attached to one or moreside chains of a derivative. In certain embodiments, PEG is used toimprove the therapeutic capacity of the interferon-associated antigenbinding protein. Certain such methods are discussed, for example, inU.S. Pat. No. 6,133,426, which is hereby incorporated by reference forany purpose.

In certain embodiments, interferon-associated antigen binding proteinvariants include glycosylation variants wherein the number and/or typeof glycosylation site has been altered compared to the amino acidsequences of a parent polypeptide. In certain embodiments, proteinvariants comprise a greater number of N-linked glycosylation sites thanthe native protein. In other embodiments, protein variants comprise alesser number of N-linked glycosylation sites than the native protein.An N-linked glycosylation site is characterized by the sequence:Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as Xcan be any amino acid residue except proline. The substitution of aminoacid residues to create this sequence provides a potential new site forthe addition of an N-linked carbohydrate chain. Alternatively,substitutions which eliminate this sequence will remove an existingN-linked carbohydrate chain. Also provided is a rearrangement ofN-linked carbohydrate chains wherein one, two, three, four, five or moreN-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated. Additional preferred variants include cysteine variants whereinone or more cysteine residues are deleted from or substituted foranother amino acid (e.g., serine) as compared to the parent amino acidsequence. Cysteine variants can be useful when antibodies must berefolded into a biologically active conformation such as after theisolation of insoluble inclusion bodies. Cysteine variants generallyhave fewer cysteine residues than the native protein, and typically havean even number to minimize interactions resulting from unpairedcysteines.

HBV and HBV Marker

As used herein, “hepatitis B virus” or “HBV” refers to the doublestranded DNA virus that causes hepatitis B, which belongs to a family ofclosely related DNA viruses called the Hepadnaviruses. Hepadnaviruseshave a strong preference for infecting liver cells, but small amounts ofhepadnaviral DNA can be found in kidney, pancreas, and mononuclearcells. However, infection at these sites is not linked to extra hepaticdisease.

The HBV virion, i.e., the Dane particle, consists of an outer lipidenvelope and an icosahedral nucleocapsid core composed of protein. Thenucleocapsid encloses the viral DNA and a DNA polymerase that hasreverse transcriptase activity similar to retroviruses. The outerenvelope contains embedded proteins, which are involved in viral bindingof, and entry into, susceptible cells. The virus is one of the smallestenveloped animal viruses with a virion diameter of 42 nm, butpleomorphic forms exist, including filamentous and spherical bodieslacking a core. These particles are not infectious and are composed ofthe lipid and protein that forms part of the surface of the virion,which is called the surface antigen (HBsAg), and is produced in excessduring the life cycle of the virus. HBV comprises HBsAg, HBcAg (and itssplice variant HBeAg), DNA polymerase and Hbx. HBV is one of a few knownnon-retroviral viruses which employ reverse transcription as a part ofits replication process.

The HBV nucleocapsid contains a relatively small and partially duplex3.2 kb circular DNA, viral polymerase and core protein. The genome hasonly four long open reading frames. The pre-S-S (pre-surface-surface)region of the genome encodes the three viral surface antigens bydifferential initiation of translation at each of three in-frameinitiation codons.

The most abundant protein of HBV is the 24 kD S protein (which is knownas HBsAg). The pre-C-C (pre-core-core) region encodes HBcAg (HBV coreAntigen) and HBeAg (HBV e Antigen). HBeAg is not required for viralreplication and plays no role in viral assembly but is nevertheless auseful indicator of active viral replication. Since HBeAg is secreted byHBV-infected hepatocytes, it can be detected in the blood via standarddiagnostic tests (such as ELISA) and is thus used as a laboratory markerfor a viremic HBV infection (Testoni et al., Serum hepatitis Bcore-related antigen (HBcrAg) correlates with covalently closed circularDNA. J. Hepatol. 2019, 70, 615-625.http://dx.doi.org/10.1016/j.jhep.2018.11.030).

The P-coding region is specific for the viral polymerase, amultifunctional enzyme involved in DNA synthesis and RNA encapsidation.The X open reading frame encodes the viral X protein (HBx), whichmodulates host-cell signal transduction and can directly and indirectlyaffect host and viral gene expression.

The life cycle of HBV is believed to begin when the virus attaches tothe host cell membrane via its envelope proteins. It has been suggestedthat HBV binds to a receptor on the plasma membrane that ispredominantly expressed on human hepatocytes via the pre-S1 domain ofthe large envelope protein as an initial step in HBV infection. However,the nature of the receptor remains controversial. Then, the viralmembrane fuses with the cell membrane and the viral genome is releasedinto the cells.

Replication of HBV can be regulated by a variety of factors, includinghormones, growth factors, and cytokines. After the viral genome reachesthe nucleus, the cellular DNA repair machinery convert the partialdouble-stranded DNA (dsDNA; also called relaxed circular HBV DNA(rcDNA)), genome into covalently closed circular DNA (cccDNA). Theresulting cccDNA is the template for host RNA Pol-II for furthertranscription of pre-genomic RNA and sub-genomic RNA (Allweiss L andDandri M, The Role of cccDNA in HBV Maintenance. Viruses 2017, 9(6):156; doi:10.3390/v9060156; Nur K. Mohd-Ismail, Zijie Lim, JayanthaGunaratne and Yee-Joo Tan, Mapping the Interactions of HBV cccDNA withHost Factors. Int. J. Mol. Sci. 2019, 20(17):4276;doi:10.3390/ijms20174276).

The pre-genomic RNA is bifunctional, serving as both the template forviral DNA synthesis and as the messenger for pre-C, C, and Ptranslation. The sub-genomic RNAs function exclusively for translationof the envelope and X protein. All viral RNA is transported to thecytoplasm, where its translation yields the viral envelope, core, andpolymerase proteins, as well as HBx and HBcAg.

HBV core particles are assembled in the cytosol and during this process,a single molecule of pre-genomic RNA is incorporated into the assemblingviral core. Once the viral RNA is encapsidated, reverse transcriptionbegins. The synthesis of the two viral DNA strands is sequential. Thefirst DNA strand is made from the encapsidated RNA template; during orafter the synthesis of this strand, the RNA template is degraded and thesynthesis of the second DNA strand proceeds, with the use of the newlymade first DNA strand as a template. Some cores bearing the maturegenome are transported back to the nucleus, where their newly minted DNAgenomes can be converted to cccDNA to maintain a stable intranuclearpool of transcriptional templates.

HBV surface antigen (HBsAg) proteins are initially synthesized andpolymerized in the rough endoplasmic reticulum. These proteins aretransported to the post-ER and pre-Golgi compartments, where budding ofthe nucleocapsid follows. The assembled HBV virion and sub-viralparticles are transported to the Golgi for further modification ofglycans of the surface proteins, and then are secreted out of the hostcell to finish the life cycle.

In particular embodiments, the interferon-associated antigen bindingproteins, the nucleic acids, vectors, vector systems, methods andcompositions described herein can be used to treat HBV infection. Asused herein, “treat HBV infection” and “treatment of HBV infection”refers to one or more of: (i) reducing HBV viral load / viral titer;(ii) reducing the transcription of cccDNA; (iii) reducing the level ofpre-genomic RNA in cells; (iv) decreasing one or more HBV-relateddisorders; and (v) decreasing one or more HBV-related symptoms in asubject.

The terms “viral load” and “viral titer” refer to the number of viralparticles in a cell, an organ or a bodily fluid such as blood or serum.Viral load or viral titer is often expressed as viral particles, orinfectious particles per mL depending on the type of assay. Today, viralload is usually measured using international units per milliliter(IU/mL). Viral load or viral titer may alternatively be determined asso-called viral genome equivalent. A higher viral burden, titer, orviral load often correlates with the severity of an active viralinfection. Accordingly, reducing the viral load or viral titercorrelates with a reduced number of infectious viral particles, e.g., inthe serum. Viral load is usually determined using nucleic acidamplification based tests (NATs or NAATss). NAT/NAAT tests utilize, forexample, PCR, (quantitative) reverse transcription polymerase chainreaction (RT-PCR or qRT-PCR), nucleic acid sequence based amplification(NASBA) or probe-based assays. Real-time PCR assays for hepatitis Bvirus DNA quantification are described, e.g., in Liu et al., Virol J 14,94 (2017) doi:10.1186/s12985-017-0759-8. Due to the ease of detection ofviral DNA using PCR, the viral load is useful in clinical settings tomonitor success during treatment. A viral load of >10,000 copies/mL(2,000 IU/mL) is a strong risk predictor of hepatocellular carcinoma,independent of HBeAg status.

The terms “patient” and “subject” are used interchangeably and includehuman and non-human animal subjects, preferably human subjects, as wellas those with formally diagnosed disorders, those without formallyrecognized disorders, those receiving medical attention, those at riskof developing the disorders, etc.

In particular embodiments, the interferon-associated antigen bindingprotein, the nucleic acids, vectors, vector systems, methods andcompositions described herein can be used to reduce the HBV viral load /viral titer in an HBV-infected cell (such as in a cell culture, in anHBV-infected organ or in an HBV-infected patient). HBV viral load /viral titer may be reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to anuntreated HBV-infected cell culture or to the same patient beforetreatment. In some embodiments, HBV viral load / viral titer is reducedby at least 20%, at least 25%, at least 30%, at least 35%, at least 40%,at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% or atleast 95%. Preferably, HBV viral load / viral titer is reduced by atleast 35%, more preferably by at least 50%. In some embodiments, viralload / viral titer is determined by PCR or qRT-PCR.

In particular embodiments, the interferon-associated antigen bindingprotein, the nucleic acids, vectors, vector systems, methods andcompositions described herein can be used to reduce transcription of HBVcccDNA in an HBV-infected cell (such as in a cell culture, in anHBV-infected organ or in an HBV-infected patient). cccDNA transcriptionmay be reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an untreatedHBV-infected cell culture or to the same patient before treatment. Insome embodiments, transcription of HBV cccDNA is reduced by at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% or at least95%. Preferably, transcription of HBV cccDNA is reduced by at least 35%,more preferably by at least 50%. In some embodiments, transcription ofHBV cccDNA is determined by PCR or qPCR.

In particular embodiments, the interferon-associated antigen bindingprotein, the nucleic acids, vectors, vector systems, methods andcompositions described herein can be used to reduce the level ofpre-genomic HBV RNA in an HBV-infected cell (such as in a cell culture,in an HBV-infected organ or in an HBV-infected patient). Pre-genomic HBVRNA levels may be reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to anuntreated HBV-infected cell culture or to the same patient beforetreatment. In some embodiments, the level of pre-genomic HBV RNA isreduced by at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90% or at least 95%. Preferably, the level of pre-genomic HBV RNAis reduced by at least 35%, more preferably by at least 50%. In someembodiments, the level of pre-genomic HBV RNA is determined by qRT-PCR.

As used herein, an “HBV-related disorder” refers to a disorder thatresults from infection of a subject by HBV. HBV-related disordersinclude, but are not limited to acute hepatitis, chronic hepatitis,icteric hepatitis, fulminant hepatitis, sub-fulminant hepatitis, andsymptoms and/or complications arising from any of these disorders.

As used herein, an “HBV-related symptom,” a “symptom of HBV infection”or an “HBV-related complication” includes one or more physicaldysfunctions related to HBV infection. HBV symptoms and complicationsinclude, but are not limited to, cirrhosis, hepatocellular carcinoma(HCC), membranous glomerulonephritis (MGN), death, acute necrotizingvasculitis (polyarteritis nodosa), membranous glomerulonephritis,papular acrodermatitis of childhood (Gianotti-Crosti syndrome),HBV-associated nephropathy (e.g., membranous glomerulonephritis),immune-mediated hematological disorders (e.g., essential mixedcryoglobulinemia, aplastic anemia), portal hypertension, ascites,encephalopathy, jaundice, pruritus, pale stools, steatorrhea,polyarteritis nodosa, glomerular disease, abnormal ALT levels, abnormalAST levels, abnormal alkaline phosphatase levels, increased bilirubinlevels, anorexia, malaise, fever, nausea, vomiting and the like.

Interferons

As used herein, an “interferon” or “IFN” refers to a cytokine, orderivative thereof, that is typically produced and released by cells inresponse to the presence of a pathogen or a tumor cell. IFNs includetype I IFNs (e.g., IFNα, IFNβ, IFNε, IFNκ, IFNτ, IFNζ and IFNω), type IIIFNs (e.g., IFNγ) and type III IFNs (e.g., IFNλ1, IFNλ2 and IFNλ3). Theterm “interferon” or “IFN” includes without limitation full-length IFN,a variant or a derivative thereof (e.g., a chemically (e.g., PEGylated)modified derivative or mutein), or a functionally active fragmentthereof, that retains one or more signaling activities of a full-lengthIFN.

As used herein, the term “functional fragment” refers to a fragment of asubstance that retains one or more functional activities of the originalsubstance. For example, a functional fragment of an interferon refers toa fragment of an interferon that retains an IFN function as describedherein, e.g., it mediates IFN pathway signaling.

The IFN may increase one or more IFN receptor activities by at leastabout 20% when added to a cell, tissue or organism expressing a cognateIFN receptor (IFNAR for IFNα, IFNBR for IFNβ, etc). In some embodiments,the interferon activates IFN receptor activity by at least 40%, at least50%, at least 60%, at least 70%, at least 80%, or at least 85%. Theactivity of the IFN (i.e., the “IFN activity”) may be measured, e.g.,using an in vitro reporter cell assay, e.g., using HEK-Blue™ IFN-α/βcells (InvivoGen, Cat. #: hkb-ifnαβ), HEK-Blue™ IFN-λ, (InvivoGen, Cat.#: hkb-ifnl) or HEK-Blue™ Dual IFN-y cells (InvivoGen, Cat. #:hkb-ifng), as described in greater detail in Example I. These reportercells were generated by stable transfection of HEK293 cells with humanIFN receptor genes and an IFN-stimulated response element-controlledsecreted embryonic alkaline phosphatase (SEAP) construct to measure theactivity of IFNs. HEK-Blue™ IFN-cells are designed to monitor theactivation of the JAK/STAT/ISGF3 pathways induced by type I, type II ortype III interferons. Activation of these pathways induces theproduction and release of SEAP.

In the context of the present invention, the interferon-associatedantigen binding proteins activate both the CD40 and an IFN pathway. Incertain embodiments, the interferon-associated antigen binding proteinactivates the IFN pathway with an EC₅₀ of less than 100, 60, 50, 40, 30,20, 10, or 1 ng/mL, preferably with an EC₅₀ of less than 11 ng/mL, morepreferably with an EC₅₀ of less than 6 ng/mL. In some of theseembodiments, the IFN pathway is the IFNα (interferon alpha), IFNβ(interferon beta), IFNε (interferon epsilon), IFNω (interferon omega),IFNγ (interferon gamma), or IFNλ (interferon lambda) pathway.

According to certain exemplary embodiments, an interferon-associatedantigen binding protein as described herein comprises full-length IFN, avariant or a derivative thereof (e.g., a chemically (e.g., PEGylated)modified derivative or mutein), or a functionally active fragmentthereof, that retains one or more signaling activities of a full-lengthIFN. In certain embodiments, the IFN is a human IFN.

In certain embodiments, an interferon-associated antigen binding proteinas described herein comprises an IFN or a functional fragment thereofselected from the group consisting of a Type I IFN, a Type II IFN and aType III IFN, or a functional fragment thereof.

In particular embodiments, the IFN or the functional fragment thereof isa Type I IFN, or a functional fragment thereof. In specific embodiments,the type I IFN or the functional fragment thereof is IFNα, IFNβ, IFNω orIFNε, or a functional fragment thereof. In more specific embodiments,the type I IFN or the functional fragment thereof is IFNα or IFNβ, or afunctional fragment thereof. In other more specific embodiments, thetype I IFN or the functional fragment thereof is IFN α, or a functionalfragment thereof. In other more specific embodiments, the type I IFN orthe functional fragment thereof is IFN β, or a functional fragmentthereof. In other more specific embodiments, the type I IFN or thefunctional fragment thereof is IFNω, or a functional fragment thereof.In other more specific embodiments, the type I IFN or the functionalfragment thereof is IFNε, or a functional fragment thereof.

In particular embodiments, the IFN or the functional fragment thereof isIFNα, IFNβ, IFNγ, IFNλ, IFNε or IFNω, or a functional fragment thereof.In specific embodiments, the IFN or a functional fragment thereof isIFNα or IFNβ, or a functional fragment thereof.

In some embodiments, the IFN or the functional fragment thereof is IFNα,or a functional fragment thereof. In more specific embodiments, the IFNor functional fragment thereof is IFNα2a, or a functional fragmentthereof. The IFNα2a may comprise the sequence as set forth in SEQ ID NO17, or a sequence at least 90% identical thereto.

In some embodiments, the IFN or the functional fragment thereof is IFNβ,or a functional fragment thereof. The IFNβ may comprise the sequence asset forth in SEQ ID NO 14, or a sequence at least 90% identical thereto.The IFNβ or the functional fragment thereof may comprise one or twoamino acid substitution(s) relative to SEQ ID NO 14, selected from C17Sand N80Q. In some embodiments, the IFNβ or the functional fragmentthereof comprises the amino acid substitution C17S relative to SEQ ID NO14. In some embodiments, the IFNβ comprises the amino acid sequence asset forth in SEQ ID NO 15. In other embodiments, the IFNβ comprises theamino acid substitutions C17S and N80Q relative to SEQ ID NO 14. In yetother embodiments, the IFNβ comprises the amino acid sequence as setforth in SEQ ID NO 16.

In some embodiments, the IFN or the functional fragment thereof is IFNγor IFNλ, or a functional fragment thereof. In specific embodiments, theIFN or functional fragment thereof is IFNγ, or a functional fragmentthereof. In more specific embodiments, the IFNγ comprises the sequenceas set forth in SEQ ID NO 19, or a sequence at least 90% identicalthereto. In other specific embodiments, the IFN or functional fragmentthereof is IFNλ, or a functional fragment thereof. In more specificembodiments, the IFNλ or the functional fragment thereof is IFNλ2, or afunctional fragment thereof. The IFNλ2 may comprise the sequence as setforth in SEQ ID NO 18, or a sequence at least 90% identical thereto.

In some embodiments, the IFN or the functional fragment thereof is IFNε,or a functional fragment thereof. The IFNε may comprise the sequence asset forth in SEQ ID NO 61, or a sequence at least 90% identical thereto.

In some embodiments, the IFN or the functional fragment thereof is IFNω,or a functional fragment thereof. The IFNω may comprise the sequence asset forth in SEQ ID NO 60, or a sequence at least 90% identical thereto.

In certain embodiments, the expression level of one or more IFNsignaling pathway biomarkers is altered, i.e., upregulated ordownregulated, in an HBV-infected cell treated with aninterferon-associated antigen binding protein described herein.According to certain exemplary embodiments, the expression level of oneor more IFN pathway biomarkers is upregulated in an HBV-infected celltreated with an interferon-associated antigen binding protein describedherein. In this context, a “biomarker” is to be understood as acharacteristic that is objectively measured and evaluated as anindicator of normal biological processes, pathogenic processes, orpharmacologic responses to a therapeutic intervention.

According to certain embodiments, a suitable IFN pathway biomarkerfeatured herein is a chemokine, e.g., a C-X-C chemokine, selected fromthe group consisting of CXCL9, CXCL10 and CXCL11. In certain exemplaryembodiments, a suitable biomarker induced by the IFN pathway is CXCL9,CXCL10 and/or CXCL11, and also the interferon stimulated gene ISG20.Cytokine induction or release may be quantified using techniques knownin the art, such as ELISA. Alternatively, induction may also bedetermined using RNA-based assays such as RNAseq or qRT-PCR. In certainembodiments, upregulation may refer to an at least at 1.5-fold, at least2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least5-fold or at least 10-fold increased expression or secretion of thesecytokines.

In these or in other exemplary embodiments, the expression level ofpro-inflammatory cytokines, e.g., IL10, IL1β and/or IL2 is notsignificantly upregulated in human Whole Blood cells upon treatment withan interferon-associated antigen binding protein of the invention. Insome embodiments, the expression level of IL10 is not significantlyupregulated in human Whole Blood cells upon treatment with aninterferon-associated antigen binding protein of the invention. In someembodiments, the expression level of IL1β is not significantlyupregulated in human Whole Blood cells upon treatment with aninterferon-associated antigen binding protein of the invention. In someembodiments, the expression level of IL2 is not significantlyupregulated in an HBV-infected cell upon treatment with aninterferon-associated antigen binding protein of the invention. In someembodiments, the expression levels of IL10 and IL1β are notsignificantly upregulated in an HBV-infected cell upon treatment with aninterferon-associated antigen binding protein of the invention. In someembodiments, the expression levels of IL10 and IL2 are not significantlyupregulated in an HBV-infected cell upon treatment with aninterferon-associated antigen binding protein of the invention. In someembodiments, the expression levels of IL1β and IL2 are not significantlyupregulated in an HBV-infected cell upon treatment with aninterferon-associated antigen binding protein of the invention. In someembodiments, the expression levels of IL10, IL1β and IL2 are notsignificantly upregulated in an HBV-infected cell upon treatment with aninterferon-associated antigen binding protein of the invention.

Interferon-Associated Antigen Binding Proteins

The term “associated”, as used herein, generally refers to a covalent ornon-covalent linkage of two (or more) molecules. Associated proteins arecreated by joining two or more distinct peptides or proteins, resultingin a protein with one or more functional properties derived from each ofthe original proteins. In the context of the present invention, theinterferon-associated antigen binding proteins activate both the CD40and an IFN pathway. An associated protein encompasses monomeric andmultimeric, e.g., dimeric, trimeric, tetrameric or the like, complexesof distinct associated or fused proteins. In this context, non-covalentlinkage results from strong interactions between two protein surfaceregions, usually via ionic, Van-der-Waals, and/or hydrogen bondinteractions. Covalent linkage, on the other hand, requires the presenceof actual chemical bonds, such as peptide bonds, disulphide bridges,etc. The term “fused” as used herein, generally refers to the joining oftwo or more distinct peptides or proteins in a covalent fashion via apeptide bond. Thus, a “fused protein” refers to single protein createdby joining two or more distinct peptides or proteins via a peptide bondwith one or more functional properties derived from each of the originalproteins. In certain embodiments, two or more distinct peptides orproteins may be fused to one another via one or more peptide linkers(“L”).

In all aspects of the invention, an interferon-associated antigenbinding protein is a protein comprising an agonistic anti-CD40 antibodyor an agonistic antigen binding fragment thereof and an IFN or afunctional fragment thereof.

In some embodiments, the IFN or the functional fragment thereof isnon-covalently associated with the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof. In more specificembodiments, the IFN or the functional fragment thereof isnon-covalently associated with the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof via ionic, Van-der-Waals,and/or hydrogen bond interactions.

In other embodiments, the IFN or the functional fragment thereof iscovalently associated with the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof. In preferred embodiments,the IFN or the functional fragment thereof is fused to the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereof.The IFN or the functional fragment thereof may be fused to a light chainof the agonistic anti-CD40 antibody or the agonistic antigen bindingfragment thereof. In some embodiments, the IFN or the functionalfragment thereof is fused to the N-terminus of a light chain of theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof. In other embodiments, the IFN or the functional fragmentthereof is fused to the C-terminus of a light chain of the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereof.The IFN or the functional fragment thereof may be also be fused to aheavy chain of the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof. In some embodiments, the IFN or the functionalfragment thereof is fused to the N-terminus of a heavy chain of theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof. In other embodiments, the IFN or the functional fragmentthereof is fused to the C-terminus of a heavy chain of the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereof. Inany of these embodiments, the agonistic anti-CD40 antibody or anagonistic antigen binding fragment thereof, and the IFN or thefunctional fragment thereof may be fused to each other via a linker.

The term “linker” or “L,” as used herein, refers to any moiety thatcovalently joins one or more agonistic anti-CD40 antibody or anagonistic antigen binding fragment thereof to one or more interferon, ora functional fragment thereof. In exemplary embodiments, a linker is apeptide linker. The term “peptide linker”, as used herein, refers to apeptide adapted to link two or more moieties. A peptide linker referredto herein may have one or more of the properties outlined in thefollowing. The sequences of peptide linker according to certainexemplary embodiments are set forth in Table 7.

A peptide linker may have any length, i.e., comprise any number of aminoacid residues. In exemplary embodiments, the linker comprises at least1, at least 2, at least 3, at least 4, at least 5 amino acids. Thelinker may comprise at least 4 amino acids. The linker may comprise atleast 11 amino acids. The linker may comprise at least 12 amino acids.The linker may comprise at least 13 amino acids. The linker may compriseat least 15 amino acids. The linker may comprise at least 20 aminoacids. The linker may comprise at least 21 amino acids. The linker maycomprise at least 24 amino acids.

A linker is typically long enough to provide an adequate degree offlexibility to prevent the linked moieties from interfering with eachother’s activity, e.g., the ability of a moiety to bind to a receptor.In exemplary embodiments, the linker comprises up to 10, up to 20, up to30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to100 amino acids. The linker may comprise up to 80 amino acids. Thelinker may comprise up to 40 amino acids. The linker may comprise up to24 amino acids. The linker may comprise up to 21 amino acids. The linkermay comprise up to 20 amino acids. The linker may comprise up to 15amino acids. The linker may comprise up to 13 amino acids. The linkermay comprise up to 12 amino acids. The linker may comprise up to 11amino acids. The linker may comprise up to 4 amino acids.

In some embodiments, the linker is selected from the group comprisingrigid, flexible and/or helix-forming linkers. It is understood thathelix-forming linkers can also be rigid linkers, since an α-helix hasless degrees of freedom than a peptide assuming a more random-coilconformation. In some embodiments, the linker is a rigid linker. Anexemplary rigid linker comprises a sequence as set forth in SEQ ID NO20. Further exemplary rigid linkers comprise a sequence as set forth inSEQ ID NO 22 or SEQ ID NO 23. In related embodiments, the linker is ahelix-forming linker. Exemplary helix-forming linkers comprise asequence as set forth in SEQ ID NO 22 or SEQ ID NO 23. In otherembodiments, the linker is a flexible linker. Exemplary flexible linkerscomprise a sequence as set forth in SEQ ID NO 21, SEQ ID NO 24, SEQ IDNO 25 or SEQ ID NO 26.

The linker can also have different chemical properties. A linker can beselected from acidic, basic or neutral linkers. Typically, acidiclinkers contain one or more acidic amino acid, such as Asp or Glu. Basiclinkers typically contain one or more basic amino acids, such as Arg,His and Lys. Both types of amino acids are very hydrophilic. In someembodiments, the linker is an acidic linker. Exemplary acidic linkerscomprise a sequence as set forth in SEQ ID NO 22 or SEQ ID NO 23. Inother embodiments, the linker is a basic linker. In yet otherembodiments, the linker is a neutral linker. Exemplary neutral linkerscomprise a sequence as set forth in SEQ ID NO 20, SEQ ID NO 21, SEQ IDNO 24, SEQ ID NO 25 or SEQ ID NO 26.

In preferred embodiments, the linker is Gly-Ser or a Gly-Ser-Thr linkercomposed of multiple glycine, serine and, where applicable, threonineresidues. In some of these embodiments, the linker comprises the aminoacids glycine and serine. In more specific embodiments, the linkercomprises the sequence as set forth in SEQ ID NO 21, SEQ ID NO 24, SEQID NO 25, SEQ ID NO 26. In some embodiments, the linker furthercomprises the amino acid threonine. In a more specific embodiment, thelinker comprises the sequence as set forth in SEQ ID NO 21.

In exemplary embodiments of the present invention, theinterferon-associated antigen binding protein comprises a linkercomprising a sequence selected from the sequences as set forth in SEQ IDNOs 20 to 26, preferably from the sequences as set forth in SEQ ID NO24, SEQ ID NO 25 or SEQ ID NO 26. In a preferred embodiment, the linkercomprises a sequence as set forth in SEQ ID NO 24. In another preferredembodiment, the linker comprises a sequence as set forth in SEQ ID NO25. In another preferred embodiment, the linker comprises a sequence asset forth in SEQ ID NO 26.

In various embodiments of any one of the aspects of the invention, theinterferon-associated antigen binding protein comprises no amino acidsother than those forming (I) said agonistic anti-CD40 antibody, oragonistic antigen binding fragment thereof and (II) said IFN orfunctional fragment thereof. In related embodiments, theinterferon-associated antigen binding protein comprises no amino acidsother than those forming (I) said agonistic anti-CD40 antibody, oragonistic antigen binding fragment thereof, (II) said IFN or functionalfragment thereof and (III) said linker.

Exemplary embodiments representing the various different configurationsof (I) the agonistic anti-CD40 antibody or the agonistic antigen bindingfragment thereof, (II) the interferon (IFN) or the functional fragmentthereof and (III) the linker are outlined in the following.

In certain preferred embodiments, the IFN or a functional fragmentthereof is fused to the C-terminus of a heavy chain of the agonisticanti-CD40 antibody, or the agonistic antigen binding fragment thereof,via the linker, as set forth in Table 3A or Table 3B. In theseembodiments, the heavy chain of the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof, may comprise a sequence asset forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48 or SEQID NO 49. The IFNα2a may comprise the sequence as set forth in SEQ ID NO17. The IFNβ may comprise the sequence as set forth in SEQ ID NO 14, SEQID NO 15 or SEQ ID NO 16. The IFNβ may comprise the sequence as setforth in SEQ ID NO 14. The IFNβ_C17S may comprise the sequence as setforth in SEQ ID NO 15. The IFNβ_C17S,N80Q may comprise the sequence asset forth in SEQ ID NO 16. The IFNγ may comprise the sequence as setforth in SEQ ID NO 19. The IFNλ2 may comprise the sequence as set forthin SEQ ID NO 18. The IFNε may comprise the sequence as set forth in SEQID NO 61. The IFNω may comprise the sequence as set forth in SEQ ID NO60. The linkers referred to are those listed in Table 7.

In the embodiments where the IFN is fused to the C-terminus of the heavychain of the agonistic anti-CD40 antibody, or the agonistic antigenbinding fragment thereof, the interferon-associated antigen bindingprotein further comprises a light chain of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof. In morespecific embodiments, a heavy chain comprises a sequence as set forth inSEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48, or SEQ ID NO 49and a light chain comprises a sequence as set forth in SEQ ID NO 3.

TABLE 3 Interferon or a functional fragment thereof fused to theC-terminus of a heavy chain of the anti-CD40 antibody or an agonisticantigen binding fragment thereof A IFNα2a IFNβ IFNβ_C17S IFNβ_C17S,N80QIFNγ IFNλ2 RL linker antiCD40_HC--RL--IFNα2a antiCD40_HC--RL--IFNβantiCD40_HC--RL--IFNβ_C17S antiCD40_HC--RL--IFNβ_C17S,N80QantiCD40-HC--RL--IFNγ antiCD40-HC--RL--IFNλ2 GST linkerantiCD40_HC--GST--IFNα2a antiCD40_HC--GST--IFNβantiCD40_HC--GST--IFNβ_C17S antiCD40_HC--GST--IFNβ_C17S,N80QantiCD40_HC--GST--IFNγ antiCD40_HC--GST--IFNλ2 HL linkerantiCD40_HC--HL--IFNα2a antiCD40_HC--HL--IFNβ antiCD40_C--HL--IFNβ_C17SantiCD40_HC--HL--IFNβ_C17S,N80Q antiCD40_HC--HL--IFNγantiCD40_HC--HL--IFNλ2 HL2 linker antiCD40_C--HL2--IFNα2aantiCD40_HC--HL2-IFNβ antiCD40_HC--HL2--IFNβ_C17SantiCD40_HC--HL2--IFNβ_C17S,N80Q antiCD40_HC--HL2--IFNγantiCD40_C--HL2--IFNλ2 (G4S)2 linker antiCD40_HC--(G4S)2--IFNα2aantiCD40_HC--(G4S)2-IFNβ antiCD40_HC--(G4S)2--IFNβ_C17SntiCD40_HC--(G4S)2--IFNβ_C17S,N80Q antiCD40_HC--(G4S)2--IFNγantiCD40_C--(G4S)2--IFNλ2 (G4S)3 linker antiCD40_HC--(G4S)3--IFNα2antiCD40_HC--(G4S)3--IFNβ antiCD40_HC--(G4S)3--IFNβ_C17SantiCD40_HC--(G4S)3--IFNβ_C17S,N80Q antiCD40_HC--(G4S)3--IFNγantiCD40_HC--(G4S)3-IFNλ2 (G4S)4 linker antiCD40_HC--(G4S)4--IFNα2aantiCD40_HC--(G4S)4-IFNβ antiCD40_HC--(G4S)4--IFNβ_C17SantiCD40_HC--(G4S)4--IFNβ_C17S,N80Q antiCD40_HC--(G4S)4--IFNγantiCD40_HC--(G4S)4--IFNλ2

B IFNε IFNω RL linker antiCD40_HC--RL--IFNε antiCD40_HC--RL--IFNω GSTlinker antiCD40_HC--GST--IFNε antiCD40_HC--GST--IFNω HL linkerantiCD40_HC--HL--IFNε antiCD40_HC--HL--IFNω HL2 linkerantiCD40_HC--HL2--IFNε antiCD40_HC--HL2--IFNω (G4S)2 linkerantiCD40_HC--(G4S)2--IFNε antiCD40_HC--(G4S)2--IFNω (G4S)3 linkerantiCD40_HC--(G4S)3--IFNε antiCD40_HC--(G4S)3--IFNω (G4S)4 linkerantiCD40_HC--(G4S)4--IFNε antiCD40_HC--(G4S)4--IFNω

In certain preferred embodiments, the IFN or a functional fragmentthereof is fused to the N-terminus of a heavy chain of the agonisticanti-CD40 antibody, or the agonistic antigen binding fragment thereof,via the linker, as set forth in Table 4A or Table 4B. In theseembodiments, the heavy chain of the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof, may comprise a sequence asset forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48, SEQID NO 49 or SEQ ID NO 50. The IFNα2a may comprise the sequence as setforth in SEQ ID NO 17. The IFNβ may comprise the sequence as set forthin SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16. The IFNβ may comprise thesequence as set forth in SEQ ID NO 14. The IFNβ_C17S may comprise thesequence as set forth in SEQ ID NO 15. The IFNβ_C17S,N80Q may comprisethe sequence as set forth in SEQ ID NO 16. The IFNγ may comprise thesequence as set forth in SEQ ID NO 19. The IFNλ2 may comprise thesequence as set forth in SEQ ID NO 18. The IFNε may comprise thesequence as set forth in SEQ ID NO 61. The IFNω may comprise thesequence as set forth in SEQ ID NO 60. The linkers referred to are thoselisted in Table 7.

In the embodiments where the IFN is fused to the N-terminus of a heavychain of the agonistic anti-CD40 antibody, or the agonistic antigenbinding fragment thereof, the interferon-associated antigen bindingprotein further comprises a light chain of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof. In morespecific embodiments, a heavy chain comprises a sequence as set forth inSEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48, SEQ ID NO 49 orSEQ ID NO 50 and a light chain comprises a sequence as set forth in SEQID NO 3.

TABLE 4 Interferon or a functional fragment thereof fused to theN-terminus of a heavy chain of the anti-CD40 antibody or an agonisticantigen binding fragment thereof A IFNα2a IFNβ IFNβ_C17S IFNβ_C17S,N80QIFNγ IFNλ2 RL linker IFNα2a--RL--antiCD40_HC IFNβ--RL--antiCD40_HCIFNβ_C17S--RL--antiCD40_HC IFNβ_C17S,N80Q--RL--antiCD40_HCIFNγ--RL--antiCD40_HC IFNλ2--RL--antiCD40_HC GST linkerIFNα2a--GST--antiCD40_HC IFNβ--GST--antiCD40_HCIFNβ_C17S--GST--antiCD40_HC IFNβ_C17S,N80Q--GST--antiCD40_HCIFNγ--GST--antiCD40_HC IFNλ2--GST--antiCD40_HC HL linkerIFNα2a--HL--antiCD40_HC IFNβ--HL--antiCD40_HC IFNβ_C17S--HL--antiCD40_HCIFNβ_C17S,N80Q--HL--antiCD40_HC IFNγ--HL--antiCD40_HCIFNλ2--HL--antiCD40_HC HL2 linker IFNα2a--HL2--antiCD40_HCIFNβ--HL2--antiCD40_HC IFNβ_C17S-HL2--antiCD40_HCIFNβ_C17S,N80Q--HL2--antiCD40_HC IFNγ--HL2--antiCD40_HCIFNλ2--HL2--antiCD40_HC (G4S)2 linker IFNα2a--(G4S)2--antiCD40_HCIFNβ_(G4S)2--antiCD40_HC IFNβ_C17S--(G4S)2--antiCD40_HCIFNβ_C17S,N80Q--(G4S)2--antiCD40_HC IFNγ--(G4S)2--antiCD40_HCIFNλ2--(G4S)2--antiCD40_HC (G4S)3 linker IFNα2a--(G4S)3--antiCD40_HCIFNβ--(G4S)3--antiCD40_HC IFNβ_C17S--(G4S)3--antiCD40_HCIFNβ_C17S,N80Q--(G4S)3--antiCD40_HC IFNγ--(G4S)3--antiCD40_HCIFNλ2--(G4S)3--antiCD40_HC (G4S)4 linker IFNα2a--(G4S)4--antiCD40_HCIFNβ--(G4S)4--antiCD40_HC IFNβ_C17S--(G4S)4--antiCD40_HCIFNβ­_C17S,N80Q--(G4S)4--antiCD40_HC IFNγ--(G4S)4--antiCD40_HCIFNλ2--(G4S)4--antiCD40_HC

B IFNε IFNω RL linker IFNε--RL--antiCD40_HC IFNω--RL--antiCD40_HC GSTlinker IFNε--GST--antiCD40_HC IFNω--GST--antiCD40_HC HL linkerIFNε--HL--antiCD40_HC IFNω--HL--antiCD40_HC HL2 linkerIFNε--HL2--antiCD40_HC IFNω--HL2--antiCD40_HC (G4S)2 linkerIFNε--(G4S)2--antiCD40_HC IFNω--(G4S)2--antiCD40_HC (G4S)3 linkerIFNε--(G4S)3--antiCD40_HC IFNω--(G4S)3--antiCD40_HC (G4S)4 linkerIFNε--(G4S)4--antiCD40_HC IFNω--(G4S)4--antiCD40_HC

In certain preferred embodiments, the IFN is fused to the C-terminus ofa light chain of the agonistic anti-CD40 antibody, or the agonisticantigen binding fragment thereof, via the linker, as set forth in Table5A or Table 5B. In these embodiments, the light chain of the agonisticanti-CD40 antibody, or the agonistic antigen binding fragment thereof,may comprise a sequence as set forth in SEQ ID NO 3. The IFNα2a maycomprise the sequence as set forth in SEQ ID NO 17. The IFNβ maycomprise the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQID NO 16. The IFNβ may comprise the sequence as set forth in SEQ ID NO14. The IFNβ_C17S may comprise the sequence as set forth in SEQ ID NO15. The IFNβ_C17S,N80Q may comprise the sequence as set forth in SEQ IDNO 16. The IFNγ may comprise the sequence as set forth in SEQ ID NO 19.The IFNλ2 may comprise the sequence as set forth in SEQ ID NO 18. TheIFNε may comprise the sequence as set forth in SEQ ID NO 61. The IFNωmay comprise the sequence as set forth in SEQ ID NO 60. The linkersreferred to are those listed in Table 7.

In the embodiments where the IFN is fused to the C-terminus of a lightchain of the agonistic anti-CD40 antibody, or the agonistic antigenbinding fragment thereof, the interferon-associated antigen bindingprotein further comprises a heavy chain of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof. In morespecific embodiments, a light chain comprises a sequence as set forth inSEQ ID NO 3 and a heavy chain comprises a sequence as set forth in SEQID NO 6, SEQ ID NO 9, SEQ ID NO 49, SEQ ID NO 48, SEQ ID NO 50 or SEQ IDNO 12.

TABLE 5 Interferon or a functional fragment thereof fused to theC-terminus of a light chain of the anti-CD40 antibody or an agonisticantigen binding fragment thereof A IFNα2a IFNβ IFNβ_C17S IFNβ_C17S,N80QIFNγ IFNλ2 RL linker antiCD40_LC— RL--IFNα2a antiCD40_LC--RL-IFNβantiCD40_LC--RL--IFNβ_C17S antiCD40_LC--RL--IFNβ_C17S,N80QantiCD40_LC--RL-IFNγ antiCD40_LC--RL--IFNλ2 GST linkerantiCD40_LC--GST--IFNα2a antiCD40_LC--GST-IFNβantiCD40_LC--GST--IFNβ_C17S antiCD40_LC--GST--IFNβ_C17S,N80QantiCD40_LC--GST--IFNγ antiCD40_LC--GST--IFNλ2 HL linkerantiCD40_LC--HL--IFNα2a antiCD40_LC--HL-IFNβ antiCD40_LC--HL— IFNβ_C17SantiCD40_LC--HL--IFNβ_C17S,N80Q antiCD40_LC--HL--IFNγantiCD40_LC--HL--IFNλ2 HL2 linker antiCD40_LC--HL2--IFNα2aantiCD40_LC--HL2-IFNβ antiCD40_LC--HL2--IFNβ_C17SantiCD40_LC--HL2--IFNβ_C17S,N80Q antiCD40_LC--HL2--IFNγantiCD40_LC--HL2-IFNλ2 (G4S)2 linker antiCD40_LC--(G4S)2--IFNα2aantiCD40_LC--(G4S)2--IFNβ antiCD40_LC--(G4S)2--IFNβ_C17SantiCD40_LC--(G4S)2--IFNβ_C17S,N80Q antiCD40_LC--(G4S)2--IFNγantiCD40_LC--(G4S)2-IFNλ2 (G4S)3 linker antiCD40_LC--(G4S)3--IFNα2aantiCD40_LC--(G4S)3--IFNβ antiCD40_LC--(G4S)3--IFNβ_C17SantiCD40_LC--(G4S)3--IFNβ_C17S,N80Q antiCD40_LC--(G4S)3--IFNγantiCD40_LC--(G4S)3-IFNλ2 (G4S)4 linker antiCD40_LC--(G4S)4--IFNα2aantiCD40_LC--(G4S)4-IFNβ antiCD40_LC--(G4S)4--IFNβ_C17SantiCD40_LC--(G4S)4--IFNβ_C17S,N80Q antiCD40_LC--(G4S)4--IFNγantiCD40_LC--(G4S)4-IFNλ2

B IFNε IFNω RL linker antiCD40_LC--RL-IFNε antiCD40_LC--RL--IFNω GSTlinker antiCD40_LC--GST-IFNε antiCD40_LC--GST--IFNω HL linkerantiCD40_LC--HL--IFNε antiCD40_LC--HL--IFNω HL2 linkerantiCD40_LC--HL2--IFNε antiCD40_LC--HL2--IFNω (G4S)2 linkerantiCD40_LC--(G4S)2--IFNε antiCD40_LC--(G4S)2--IFNω (G4S)3 linkerantiCD40_LC--(G4S)3--IFNε antiCD40_LC--(G4S)3--IFNω (G4S)4 linkerantiCD40_LC--(G4S)4-IFNε antiCD40_LC--(G4S)4--IFNω

In certain preferred embodiments, the IFN is fused to the N-terminus ofa light chain of the agonistic anti-CD40 antibody, or the agonisticantigen binding fragment thereof, via the linker, as set forth in Table6A or Table 6B. In these embodiments, the light chain of the agonisticanti-CD40 antibody, or the agonistic antigen binding fragment thereof,may comprise a sequence as set forth in SEQ ID NO 3. The IFNα2a maycomprise the sequence as set forth in SEQ ID NO 17. The IFNβ maycomprise the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQID NO 16. The IFNβ may comprise the sequence as set forth in SEQ ID NO14. The IFNβ_C17S may comprise the sequence as set forth in SEQ ID NO15. The IFNβ_C17S,N80Q may comprise the sequence as set forth in SEQ IDNO 16. The IFNγ may comprise the sequence as set forth in SEQ ID NO 19.The IFNλ2 may comprise the sequence as set forth in SEQ ID NO 18. TheIFNε may comprise the sequence as set forth in SEQ ID NO 61. The IFNωmay comprise the sequence as set forth in SEQ ID NO 60. The linkersreferred to are those listed in Table 7.

In the embodiments where the IFN is fused to the N-terminus of a lightchain of the agonistic anti-CD40 antibody, or the agonistic antigenbinding fragment thereof, the interferon-associated antigen bindingprotein further comprises a heavy chain of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof. In morespecific embodiments, a light chain comprises a sequence as set forth inSEQ ID NO 3 and a heavy chain comprises a sequence as set forth in SEQID NO 6, SEQ ID NO 9, SEQ ID NO 49, SEQ ID NO 48, SEQ ID NO 12 or SEQ IDNO 50.

TABLE 6 Interferon or a functional fragment thereof fused to theN-terminus of a light chain of the anti-CD40 antibody or an agonisticantigen binding fragment thereof A IFNα2a IFNβ IFNβ_C17S IFNβ_C17S,N80QIFNγ IFNλ2 RL linker IFNα2a--RL--antiCD40_LC IFNβ--RL-antiCD40_LCIFNβ_C17S--RL--antiCD40_LC IFNβ_C17S,N80Q--RL--antiCD40_LCIFNγ--RL--antiCD40_LC IFNλ2--RL--antiCD40_LC GST linkerIFNα2a--GST--antiCD40_LC IFNβ--GST--antiCD40_LCIFNββ_C17S--GST-antiCD40_LC IFNβ_C17S,N80Q--GST--antiCD40_LCIFNγ--GST--antiCD40_LC IFNλ2--GST--antiCD40_LC HL linkerIFNα2a--HL--antiCD40_LC IFNβ--HL--antiCD40_LC IFNβ_C17S--HL--antiCD40_LCIFNβ_C17S,N80Q--HL--antiCD40_LC IFNγ--HL--antiCD40_LCIFNλ2--HL--antiCD40_LC HL2 linker IFNα2a--HL2--antiCD40_LCIFNβ--HL2--antiCD40_LC IFNβ_C17S-HL2-antiCD40_LCIFNβ_C17S,N80Q--HL2--antiCD40_LC IFNγ--HL2--antiCD40_LCIFNλ2--HL2--antiCD40_LC (G4S)2 linker IFNα2a--(G4S)2--antiCD40_LCIFNβ--(G4S)2-antiCD40_LC IFNβ_C17S-(G4S)2-antiCD40_LCIFNβ_C17S,N80Q--(G4S)2-antiCD40_LC IFNγ--(G4S)2--antiCD40_LCIFNλ2--(G4S)2--antiCD40_LC (G4S)3 linker IFNα2a--(G4S)3--antiCD40_LCIFNβ-(G4S)3-antiCD40_LC IFNβ_C17S-(G4S)3--antiCD40_LCIFNβ_C17S,N80Q--(G4S)3--antiCD40_LC IFNγ--(G4S)3--antiCD40_LCIFNλ2--(G4S)3--antiCD40_LC (G4S)4 linker IFNα2a--(G4S)4-antiCD40_LCIFNβ-(G4S)4-antiCD40_LC IFNβ_C17S--(G4S)4-antiCD40_LCIFNβ_C17S,N80Q--(G4S)4-antiCD40_LC IFNγ--(G4S)4--antiCD40_LCIFNλ2--(G4S)4--antiCD40_LC

B IFNε IFNω RL linker IFNε--RL--antiCD40_LC IFNω--RL--antiCD40_LC GSTlinker IFNε--GST--antiCD40_LC IFNω--GST--antiCD40_LC HL linkerIFNε--HL--antiCD40_LC IFNω--HL--antiCD40_LC HL2 linkerIFNε--HL2--antiCD40_LC IFNω--HL2--antiCD40_LC (G4S)2 linkerIFNε-(G4S)2-antiCD40_LC IFNω--(G4S)2--antiCD40_LC (G4S)3 linkerIFNε--(G4S)3--antiCD40_LC IFNω--(G4S)3--antiCD40_LC (G4S)4 linkerIFNε-(G4S)4-antiCD40_LC IFNω--(G4S)4--antiCD40_LC

Exemplary sequences comprised in interferon-associated antigen bindingproteins of the invention or precursors thereof are listed in Table 7.

In exemplary preferred embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic antigen binding fragmentthereof comprising a sequence selected from SEQ ID NOs 28-47. In otherexemplary embodiments, the interferon-associated antigen binding proteincomprises an interferon-fused agonistic antiCD40 antibody or aninterferon-fused agonistic antigen binding fragment thereof comprising asequence selected from SEQ ID NOs 62-69. In exemplary preferredembodiments, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic antigen binding fragment thereof comprising a sequenceselected from SEQ ID NOs 28-47. In other exemplary embodiments, theinterferon-associated antigen binding protein is an interferon-fusedagonistic anti-CD40 antibody or an interferon-fused agonistic antigenbinding fragment thereof comprising a sequence selected from SEQ ID NOs62-69.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 62. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 62.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 63. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 63.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 64. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 64.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 65. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 65.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 66. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 66.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 67. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 67.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 68. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 68.

In certain exemplary embodiments, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 69. In another exemplaryembodiment, the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic binding fragment thereof comprising a sequence as set forth inSEQ ID NO 69.

In more preferred embodiments, the interferon-associated antigen bindingprotein comprises an interferon-fused agonistic anti-CD40 antibody or aninterferon-fused agonistic antigen binding fragment thereof comprising asequence selected from SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ IDNO 41, SEQ ID NO 42 or SEQ ID NO 43. In more preferred embodiments, theinterferon-associated antigen binding protein is an interferon-fusedagonistic anti-CD40 antibody or an interferon-fused agonistic antigenbinding fragment thereof comprising a sequence selected from SEQ ID NO38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42 or SEQ ID NO43.

In an even more preferred embodiment, the interferon-associated antigenbinding protein comprises an interferon-fused agonistic anti-CD40antibody or an interferon-fused agonistic binding fragment thereofcomprising a sequence as set forth in SEQ ID NO 38. In still anothereven more preferred embodiment, the interferon-associated antigenbinding protein is an interferon-fused agonistic antiCD40 antibody or aninterferon-fused agonistic binding fragment thereof comprising asequence as set forth in SEQ ID NO 38.

In another even more preferred embodiment, the interferon-associatedantigen binding protein comprises an interferon-fused agonisticanti-CD40 antibody or an interferon-fused agonistic binding fragmentthereof comprising a sequence as set forth in SEQ ID NO 39. In anothereven more preferred embodiment, the interferon-associated antigenbinding protein is an interferon-fused agonistic antiCD40 antibody or aninterferon-fused agonistic binding fragment thereof comprising asequence as set forth in SEQ ID NO 39.

In another even more preferred embodiment, the interferon-associatedantigen binding protein comprises an interferon-fused agonisticanti-CD40 antibody or an interferon-fused agonistic binding fragmentthereof comprising a sequence as set forth in SEQ ID NO 40. In anothereven more preferred embodiment, the interferon-associated antigenbinding protein is an interferon-fused agonistic antiCD40 antibody or aninterferon-fused agonistic binding fragment thereof comprising asequence as set forth in SEQ ID NO 40.

In another even more preferred embodiment, the interferon-associatedantigen binding protein comprises an interferon-fused agonisticanti-CD40 antibody or an interferon-fused agonistic binding fragmentthereof comprising a sequence as set forth in SEQ ID NO 41. In anothereven more preferred embodiment, the interferon-associated antigenbinding protein is an interferon-fused agonistic antiCD40 antibody or aninterferon-fused agonistic binding fragment thereof comprising asequence as set forth in SEQ ID NO 41.

In another even more preferred embodiment, the interferon-associatedantigen binding protein comprises an interferon-fused agonisticanti-CD40 antibody or an interferon-fused agonistic binding fragmentthereof comprising a sequence as set forth in SEQ ID NO 42. In anothereven more preferred embodiment, the interferon-associated antigenbinding protein is an interferon-fused agonistic antiCD40 antibody or aninterferon-fused agonistic binding fragment thereof comprising asequence as set forth in SEQ ID NO 42.

In another even more preferred embodiment, the interferon-associatedantigen binding protein comprises an interferon-fused agonisticanti-CD40 antibody or an interferon-fused agonistic binding fragmentthereof comprising a sequence as set forth in SEQ ID NO 43. In anothereven more preferred embodiment, the interferon-associated antigenbinding protein is an interferon-fused agonistic antiCD40 antibody or aninterferon-fused agonistic binding fragment thereof comprising asequence as set forth in SEQ ID NO 43.

TABLE 7 Sequences of exemplary interferon-associated antigen bindingprotein and components thereof based on the antiCD40 antibody CP870,893.Italic sequences correspond to signal peptides. Bold italic sequences inSEQ ID NOs 3 and 6 correspond to CDR regions. Bold non-italic sequencescorrespond to linkers. Mutated amino acids are underlined Name / SEQ IDNumber Sequence Signal peptide 1 (SEQ ID NO 1) MGWSCIILFLVATATGVHSSignal peptide 2 (SEQ ID NO 2) MDMRVPAQLLGLLLLWLRGARC antiCD40 antibodylight chain (SEQ ID NO 3) DIQMTQSPSSVSASVGDRVTITC RASQGIYSWLAWYQQKPGKAPNLLIY TASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECantiCD40 antibody light chain with signal peptide 1 (SEQ ID NO 4)MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECantiCD40 antibody light chain with signal peptide 2 (SEQ ID NO 5)MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECantiCD40 antibody heavy chain hIgG2 dK (SEQ ID NO 6)QVQLVQSGAEVKKPGASVKVSCKASGYTF TGYYMH WVRQAPGQGLEWMG WINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGantiCD40 antibody heavy chain hIgG2 dK with signal peptide 1 (SEQ ID NO7)MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGantiCD40 antibody heavy chain hIgG2 dK with signal peptide 2 (SEQ ID NO8)MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGantiCD40 antibody heavy chain hIgG2 (SEQ ID NO 9)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKantiCD40 antibody heavy chain hIgG2 with signal peptide 1 (SEQ ID NO 10)MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKantiCD40 antibody heavy chain hIgG2 with signal peptide 2 (SEQ ID NO 11)MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKantiCD40 antibody hIgG1 heavy chain - NNAS (SEQ ID NO 48)QVQLVQSGAEVKKPGASVKVSCKASGYTF TGYYMH WVRQAPGQGLEWMG WINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKantiCD40 antibody hIgG1 heavy chain - NNAS-dK (SEQ ID NO 49)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGantiCD40 antibody hIgG2 Fab region heavy chain (SEQ ID NO 12)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEantiCD40 antibody hIgG2 Fab region heavy chain with signal peptide 1(SEQ ID NO 13)MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEantiCD40 antibody hIgG2 Fab region heavy chain --TEV--6His tag (SEQ IDNO 50)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEENLYFQSHHHHHHIFNβ (SEQ ID NO 14)MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNIFNβ C17S (SEQ ID NO 15)MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNIFNβ C17S,N80Q (SEQ ID NO 16)MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWQETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNIFNα2a (SEQ ID NO 17)CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEIFNλ2 (SEQ ID NO 18)VPVARLHGALPDARGCHIAQFKSLSPQELQAFKRAKDALEESLLLKDCRCHSRLFPRTWDLRQLQVRERPMALEAELALTLKVLEATADTDPALVDVLDQPLHTLHHILSQFRACIQPQPTAGPRTRGRLHHWLYRLQEAPKKESPGCLEASVTFNLFRLLTRDLNCVASGDLCVIFNγ (SEQ ID NO 19)QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQIFNω (SEQ ID NO 60)LGCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSSIFNε (SEQ ID NO 61)LDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHRKNFLLPQKSLSPQQYQKGHTLAILHEMLQQIFSLFRANISLDGWEENHTEKFLIQLHQQLEYLEALMGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAIVQVEISRCLFFVFSLTEKLSKQGRPLNDMKQELTTEFRSPRRL linker (SEQ ID NO 20) PAPA GST linker (SEQ ID NO 21) SGGTSGSTSGTGS HLlinker (SEQ ID NO 22) AEAAAKEAAAKA HL2 linker (SEQ ID NO 23)AEAAAKEAAAKAAEAAAKEAAAKA (G4S)2 linker (SEQ ID NO 24) GGGGSGGGGS (G4S)3linker (SEQ ID NO 25) GGGGSGGGGSGGGGS (G4S)4 linker (SEQ ID NO 26)GGGGSGGGGSGGGGSGGGGS TEV-6His tag (SEQ ID NO 27) ENLYFQSHHHHHHantiCD40_LC--HL--IFNβ (SEQ ID NO 28)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAEAAAKEAAAKAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_LC--HL--IFNβ_C17S (SEQ ID NO 29)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAEAAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_hIgG2_ dK HC--RL--IFNβ (SEQ ID NO 30)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAPAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_hIgG2_ dK_HC--RL--IFNβ_C17S (SEQ ID NO 31)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAPAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_hIgG2_ dK_HC--HL--IFNβ (SEQ ID NO 32)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAEAAAKEAAAKAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_hIgG2_ dK_HC--HL--IFNβ_C17S (SEQ ID NO 33)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAEAAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_LC--RL--IFNβ (SEQ ID NO 34)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECPAPAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_LC--RL--IFNβ_C17S (SEQ ID NO 35)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECPAPAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_LC--GST--IFNβ_C17S (SEQ ID NO 36)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSGGTSGSTSGTGSMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_LC--HL2--IFNβ_C17S (SEQ ID NO 37)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAEAAAKEAAAKAAEAAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_hIgG2_ dK_HC--(G4S)2--IFNα2a (SEQ ID NO 38)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLSFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEantiCD40_hIgG2_ dK HC--(G4S)3--IFNα2a (SEQ ID NO 39)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEantiCD40_hIgG2_ dK_HC--(G4S)4--IFNα2a (SEQ ID NO 40)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEantiCD40_LC--(G4S)2--IFNα2a (SEQ ID NO 41)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEantiCD40_LC--(G4S)3--IFNα2a (SEQ ID NO 42)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEantiCD40_LC--(G4S)4--IFNα2a (SEQ ID NO 43)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEIFNβ--(G4S)3--antiCD40_LC) (SEQ ID NO 44)MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECantiCD40_LC--(G4S)4--IFN (SEQ ID NO 45)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNIFNβ--(G4S)3--antiCD40_HC_Ig G1 NNAS-dK (SEQ ID NO 46)MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGantiCD40_HC_Ig G1_NNAS_dK--(G4S)4--IFNβ (SEQ ID NO 47)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNantiCD40_hIgG2 dK_HC--HL--IFNα2A (SEQ ID NO 62)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAEAAAKEAAAKACDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEantiCD40_LC-derivative--HL--IFNα2A (SEQ ID NO 63)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEKSLSLSPGAEAAAKEAAAKACDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEantiCD40_LC--(G4S)4--IFNγ (SEQ ID NO 64)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQantiCD40_hIgG2 dK_HC--(G4S)4--IFNγ (SEQ ID NO 65)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQantiCD40_LC--(G4S)4--IFNλ2 (SEQ ID NO 66)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSVPVARLHGALPDARGCHIAQFKSLSPQELQAFKRAKDALEESLLLKDCRCHSRLFPRTWDLRQLQVRERPMALEAELALTLKVLEATADTDPALVDVLDQPLHTLHHILSQFRACIQPQPTAGPRTRGRLHHWLYRLQEAPKKESPGCLEASVTFNLFRLLTRDLNCVASGDLCVantiCD40_hIgG2 dK_HC--(G4S)4--IFNλ2 (SEQ ID NO 67)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSVPVARLHGALPDARGCHIAQFKSLSPQELQAFKRAKDALEESLLLKDCRCHSRLFPRTWDLRQLQVRERPMALEAELALTLKVLEATADTDPALVDVLDQPLHTLHHILSQFRACIQPQPTAGPRTRGRLHHWLYRLQEAPKKESPGCLEASVTFNLFRLLTRDLNCVASGDLCVantiCD40_LC--(G4S)4--IFNω (SEQ ID NO 68)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSLGCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSSantiCD40_hIgG2 dK_HC--(G4S)4--IFNε (SEQ ID NO 69)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSLDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHRKNFLLPQKSLSPQQYQKGHTLAILHEMLQQIFSLFRANISLDGWEENHTEKFLIQLHQQLEYLEALMGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAIVQVEISRCLFFVFSLTEKLSKQGRPLNDMKQELTTEFRSPR

In preferred embodiments, the interferon-associated antigen bindingproteins described herein are interferon-fused antigen binding proteinscomprising polypeptides derived from those specified in Table 8, inparticular Table 8A or Table 8B, more particularly Table 8A below, andespecially from the polypeptides of SEQ ID NO 38, SEQ ID NO 39, SEQ IDNO 40, SEQ ID NO 41, SEQ ID NO 42 or SEQ ID NO 43 above. In preferredembodiments, the interferon-associated antigen binding proteinsdescribed herein are interferon-fused antigen binding proteinsconsisting of polypeptides derived from those specified in Table 8, inparticular Table 8A or Table 8B, more particularly Table 8A below, andespecially from the polypeptides of SEQ ID NO 38, SEQ ID NO 39, SEQ IDNO 40, SEQ ID NO 41, SEQ ID NO 42 or SEQ ID NO 43 above. In morepreferred embodiments, the interferon-fused antibody comprises thesequences as set forth in SEQ ID NO 38 and SEQ ID NO 3. In other morepreferred embodiments, the interferon-fused antibody comprises thesequences as set forth in SEQ ID NO 39 and SEQ ID NO 3. In other morepreferred embodiments, the interferon-fused antibody comprises thesequences as set forth in SEQ ID NO 40 and SEQ ID NO 3. In other morepreferred embodiments, the interferon-fused antibody comprises thesequences as set forth in SEQ ID NO 41 and SEQ ID NO 9. In other morepreferred embodiments, the interferon-fused antibody comprises thesequences as set forth in SEQ ID NO 42 and SEQ ID NO 9. In other morepreferred embodiments, the interferon-fused antibody comprises thesequences as set forth in SEQ ID NO 43 and SEQ ID NO 9.A

TABLE 8 Polypeptide combinations found in preferred interferon-fusedantigen binding proteins of the invention, their mean EC₅₀ values withregard to the activation of CD40 andIFN-pathways and their productivity(i.e., yield per liter culture). Each sequencecombination as indicatedis comprised twice in the respective IFA. SN: supernatantInterferon-fused Antibody (IFA) Sequence combination CD40 EC₅₀ (ng/mL)IFNβ EC₅₀ (ng/mL) IFNαEC₅₀ (ng/mL) productivity (mg/L) IFA1 (SEQ ID NO28) + (SEQ ID NO 9) 74.1 1.64 16.7 IFA2 (SEQ ID NO 29) + (SEQ ID NO 9)111 0.14 17.8 IFA8 (SEQ ID NO 30) + (SEQ ID NO 3) 39.7 2.9 6.45 IFA9(SEQ ID NO 31) + (SEQ ID NO 3) 42.6 0.7 3.4 IFA10 (SEQ ID NO 32) + (SEQID NO 3) 26.5 4.5 6.9 IFA11 (SEQ ID NO 33) + (SEQ ID NO 3) 42.8 1.78 5.1IFA12 (SEQ ID NO 34) + (SEQ ID NO 9) 105 3.64 21.2 IFA13 (SEQ ID NO35) + (SEQ ID NO 9) 192 0.7 11.5 IFA19 (SEQ ID NO 36) + (SEQ ID NO 9)110 1.3 5.6 IFA20 (SEQ ID NO 37) + (SEQ ID NO 9) 182 2.34 4.2 IFA25 (SEQID NO 38) + (SEQ ID NO 3) 13.3 5.1 21 IFA26 (SEQ ID NO 39) + (SEQ ID NO3) 15.35 4 8.6 IFA27 (SEQ ID NO 40) + (SEQ ID NO 3) 17 2.4 9.3 IFA28(SEQ ID NO 41) + (SEQ ID NO 9) 12.8 4.5 75 IFA29 (SEQ ID NO 42) + (SEQID NO 9) 11.1 2 56.6 IFA30 (SEQ ID NO 43) + (SEQ ID NO 9) 11.3 1.6 46.6IFA34 (SEQ ID NO 44) + (SEQ ID NO 49) active (SN) active (SN) nosignificant production IFA35 (SEQ ID NO 45) + (SEQ ID NO 49) active (SN)active (SN) no significant production IFA36 (SEQ ID NO 46) + (SEQ ID NO3) active (SN) active (SN) no significant production IFA37 (SEQ ID NO47) + (SEQ ID NO 3) active (SN) active (SN) no significant production

B

Interferon- fused Antibody (IFA) Sequence combination CD40 EC₅₀ (ng/mL)IFNα EC₅₀ (ng/mL) IFNλ EC₅₀ (ng/mL) IFNγ EC₅₀ (ng/mL) IFNε EC₅₀ (ng/mL)IFNω EC₅₀ (ng/mL) productivity (mg/L) IFA38 (SEQ ID NO 62) + (SEQ ID NO3) 22.7 3.77 1.32 IFA39 (SEQ ID NO 63) + (SEQ ID NO 9) 17.5 2.95 1.25IFA42 (SEQ ID NO 64) + (SEQ ID NO 9) 65.6 15.4 0.72 IFA43 (SEQ ID NO65) + (SEQ ID NO 3) 50.8 <0.001 0.55 IFA44 (SEQ ID NO 66) + (SEQ ID NO9) 41.4 0.153 0.91 IFA45 (SEQ ID NO 67) + (SEQ ID NO 3) 25.8 <0.001 1.09IFA46 (SEQ ID NO 68) + (SEQ ID NO 9) 86.3 0.493 0.89 IFA49 (SEQ ID NO69) + (SEQ ID NO 3) 65.8 78.2 0.61 IFA50 (SEQ ID NO 41) + (SEQ ID NO 50)128 1.36 0.57 IFA51 (SEQ ID NO 42) + (SEQ ID NO 50) 123 1.43 0.48

Nucleic Acids and Expression Vectors

In one aspect, a combination of polynucleotides encoding aninterferon-associated antigen binding protein is provided. Methods ofmaking an interferon-associated antigen binding protein comprisingexpressing these polynucleotides are also provided.

In some embodiments, a nucleic acid encoding an IFN or a functionalfragment thereof being fused to an agonistic anti-CD40 antibody or anagonistic antigen binding fragment thereof, as disclosed herein isprovided. In certain exemplary embodiments, the nucleic acid is encodingan IFN or a functional fragment thereof fused to an agonistic anti-CD40antibody or an agonistic antigen binding fragment thereof according toany of the sequences set forth in SEQ ID NOs 62 to 69, or a nucleic acidsequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to a nucleic acid encoding any ofthese sequences. In certain exemplary embodiments, said nucleic acid isat least 95%, at least 98% or at least 99% identical to a nucleic acidencoding any of SEQ ID NOs 62 to 69. In preferred embodiments, thenucleic acid is encoding an IFN or a functional fragment thereof fusedto an agonistic anti-CD40 antibody or an agonistic antigen bindingfragment thereof according to any of the sequences set forth in SEQ IDNOs 28 to 47, or a nucleic acid sequence at least 80%, at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identical to anucleic acid encoding any of these sequences. In even more specificembodiments, said nucleic acid is at least 95%, at least 98% or at least99% identical to a nucleic acid encoding any of SEQ ID NOs 28 to 47.

In those embodiments wherein a nucleic acid encodes an IFN or afunctional fragment thereof being fused to a light chain of theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof, the nucleic acid may further encode a heavy chain of theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof. In more specific embodiments, the heavy chain of the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereofcomprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13,SEQ ID NO 48, SEQ ID NO 49, or SEQ ID NO 50, or a nucleic acid sequenceat least 80%, at least 85%, at least 90%, at least 95%, at least 98% orat least 99% identical to a nucleic acid encoding any of thesesequences. In even more specific embodiments, said nucleic acid is atleast 95%, at least 98% or at least 99% identical to a nucleic acidencoding SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 48, SEQ ID NO49, or SEQ ID NO 50.

In those embodiments where a nucleic acid encodes an IFN or a functionalfragment thereof being fused to the heavy chain of the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereof,the nucleic acid may further encode a light chain of the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereof. Inmore specific embodiments, the light chain of the agonistic anti-CD40antibody or the agonistic antigen binding fragment thereof comprises asequence as set forth in SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5, or anucleic acid sequence at least 80%, at least 85%, at least 90%, at least95%, at least 98% or at least 99% identical to a nucleic acid encodingany of these sequences. In even more specific embodiments, said nucleicacid is at least 95%, at least 98% or at least 99% identical to anucleic acid encoding SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5.

In certain embodiments, the nucleic acids described herein may comprisea sequence encoding a sequence to increase the yield (e.g. a solubilitytag) or facilitate purification of the expressed proteins (i.e., apurification tag). Purification tags are known to a person skilled inthe art and may be selected from glutathione S-transferase (GST) tags,maltose binding protein (MBP) tags, calmodulin binding peptide (CBP)tags, intein-chitin binding domain (intein-CBD) tags,Streptavidin/Biotin-based tags (such as biotinylation signal peptide(BCCP) tags, Streptavidin-binding peptide (SBP) tags, His-patchThioFusion tags, tandem affinity purification (TAP) tags, Smallubiquitin-like modifier (SUMO) tags, HaloTag® (Promega), ProfinityeXact™ system (Bio-Rad). In some embodiments, the purification tag maybe a polyhistidine tag (e.g., a His₆-, His₇-, His₈-, His₉- orHis₁₀-tag). In other embodiments, the purification tag may be aStrep-tag (e.g., a Strep-tag® or a Strep-tag II®; IBA Life Sciences). Inyet other embodiments, the purification tag may be a maltose bindingprotein (MBP) tag.

In some embodiments, the nucleic acid sequence may further comprise asequence encoding a cleavage site for removal of the purification tag.Such cleavage sequences are known to a person skilled in the art and maybe selected from a sequence recognized and cleaved by an endoprotease oran exoprotease. In some embodiments, an endoprotease for the removal ofa purification tag may be selected from: Enteropeptidase, Thrombin,Factor Xa, TEV protease or Rhinovirus 3C protease. In some embodiments,an exoprotease for the removal of a purification tag may be selectedfrom: Carboxypeptidase A, Carboxypeptidase B or DAPase. In preferredembodiments, the protease for the removal of a purification tag is TEVprotease. In a more specific preferred embodiment, the nucleic acidcomprises a sequence encoding a His₆-tag and a TEV cleavage site. In aneven more specific preferred embodiment, said nucleic acid comprises asequence encoding a sequence as set forth in SEQ ID NO 27.

The nucleic acid molecules of the invention may also comprise a sequenceencoding a signal peptide. The skilled person is aware of the varioussignal peptides available to direct the expressed protein to the desiredsite of folding, assembly and/or maturation as well as to effectsecretion of the final protein into the medium to facilitate downstreamprocessing. Thus, in some embodiments, the signal peptide is a secretorysignal peptide. The encoded signal peptide may comprise a sequence asset forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, thesignal peptide comprises the sequence as set forth in SEQ ID NO: 1. Inother embodiments, the signal peptide comprises the sequence as setforth in SEQ ID NO: 2.

Signal peptide 1 (SEQ ID NO 1) was used for synthesis of the polypeptidesequences as set forth in SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID36, SEQ ID NO 37, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47or SEQ ID NO 50. Such signal peptide that is initially present at theN-terminus of the respective sequence of the polypeptide is cleavedduring synthesis.

Signal peptide 2 (SEQ ID NO 2) was used for synthesis of the polypeptidesequences as set forth in SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQID NO 41, SEQ ID NO 42 and SEQ ID NO 43. Such signal peptide that isinitially present at the N-terminus of the respective sequence of thepolypeptide is cleaved during synthesis.

For the synthesis of the polypeptide sequences as set forth in SEQ ID NO62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO67, SEQ ID 68 and SEQ ID NO 69 the signal peptide MGWSCIILFLVATATGVHS(SEQ ID NO 1) was used. Such signal peptide that is initially present atthe N-terminus of the respective sequence of the polypeptide is cleavedduring synthesis.

Polynucleotides encoding an IFN or a functional fragment thereof beingfused to the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof as disclosed herein are typically inserted inan expression vector for introduction into host cells that may be usedto produce the desired quantity of the claimed interferon-associatedantigen binding proteins. Accordingly, in certain aspects, the inventionprovides expression vectors comprising polynucleotides disclosed hereinand host cells comprising these vectors and polynucleotides.

The term “vector” or “expression vector” is used herein for the purposesof the specification and claims, to mean vectors used in accordance withthe present invention as a vehicle for introducing into and expressing adesired gene in a cell. As known to those skilled in the art, suchvectors may easily be selected from the group consisting of plasmids,phages, viruses and retroviruses. In general, vectors compatible withthe present invention will comprise a selection marker, appropriaterestriction sites to facilitate cloning of the desired gene and theability to enter and/or replicate in eukaryotic or prokaryotic cells.

Numerous expression vector systems may be employed for the purposes ofthis invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV), or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by co-transformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals. In some embodiments the clonedvariable region genes, one of them fused with a gene encoding an IFN ora functional fragment thereof, are inserted into an expression vectoralong with the heavy and light chain constant region genes (such ashuman genes) synthesized as discussed above.

In other embodiments, a vector system of the invention may comprise morethan one vector. In some embodiments, a vector system may comprise afirst vector for the expression of an IFN or a functional fragmentthereof fused to a light chain of the agonistic anti-CD40 antibody orthe agonistic antigen binding fragment thereof and a second vector forexpression of a heavy chain of the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof. Alternatively, such a vectorsystem may comprise a first vector for the expression of an IFN or afunctional fragment thereof fused to a heavy chain of the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereof anda second vector for expression of a light chain of the agonisticanti-CD40 antibody or the agonistic antigen binding fragment thereof.

In other embodiments, an interferon-associated antigen binding proteinas described herein may be expressed using polycistronic constructs. Insuch expression systems, multiple gene products of interest such asthose encoding an IFN or a functional fragment thereof being fused to aheavy chain of an agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof and encoding a light chain of said antibody, orthose encoding an IFN or a functional fragment thereof being fused to alight chain of an agonistic anti-CD40 antibody or an agonistic antigenbinding fragment thereof and encoding a heavy chain of said antibody oran agonistic antigen binding fragment thereof may be produced from asingle polycistronic construct. These systems advantageously use aninternal ribosome entry site (IRES) to provide relatively high levels ofpolypeptides in eukaryotic host cells. Compatible IRES sequences aredisclosed in U.S. Pat. No. 6,193,980, which is incorporated by referenceherein. Those skilled in the art will appreciate that such expressionsystems may be used to effectively produce the full range ofpolypeptides disclosed in the instant application.

More generally, once a vector or a DNA sequence encoding aninterferon-associated antigen binding protein of the present inventionhas been prepared, the expression vector may be introduced into anappropriate host cell. That is, the host cell may be transformed.Introduction of a plasmid into the host cell can be accomplished byvarious techniques well known to those of skill in the art. Theseinclude, but are not limited to, transfection (including electrophoresisand electroporation), protoplast fusion, calcium phosphateprecipitation, cell fusion with enveloped DNA, microinjection, andinfection with intact virus. See, e.g., Ridgway, A. A. G. “MammalianExpression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez andDenhardt, Eds. (Butterworths, Boston, MA 1988). The transformed cellsare grown under conditions appropriate to the production of the lightchains and heavy chains, and assayed for heavy and/or light chainprotein synthesis. Exemplary assay techniques include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

As used herein, the term “transformation” shall be used in a broad senseto refer to the introduction of DNA into a recipient host cell thatchanges the genotype and consequently results in a change in therecipient cell.

Along those same lines, “host cells” refer to cells that have beentransformed with vectors constructed using recombinant DNA techniquesand encoding at least one heterologous gene. In descriptions ofprocesses for isolation of polypeptides from recombinant hosts, theterms “cell” and “cell culture” are used interchangeably to denote thesource of the interferon-associated antigen binding protein unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

In one embodiment, the host cell line used for expression of aninterferon-associated antigen binding protein is of eukaryotic orprokaryotic origin. As used herein, the term “expression” may includethe transcription and translation of more than one polypeptide chain(such as a heavy and a light chain of the antibody moiety of aninterferon-associated antigen binding protein), which associate to formthe final interferon-associated antigen binding protein. In oneembodiment, the host cell line used for expression of aninterferon-associated antigen binding protein is of bacterial origin. Inone embodiment, the host cell line used for expression of aninterferon-associated antigen binding protein is of mammalian origin;those skilled in the art can determine particular host cell lines whichare best suited for the desired gene product to be expressed therein.Exemplary host cell lines include, but are not limited to, CHO K1 GSknockout from Horizon, DG44 and DUXB11 (Chinese Hamster Ovary lines,DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line),COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI(human lymphocyte), HEK 293 (human kidney). In a preferred embodiment,HEK FS S11/ 254 cells may be used. In another preferred embodiment, CHOK1 GS from Horizon may be used. In one embodiment, the cell lineprovides for altered glycosylation, e.g., afucosylation, of the antibodyexpressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-out CHO celllines (POTELLIGENT™ cells) (Biowa, Princeton, NJ)). In one embodimentNS0 cells may be used. Host cell lines are typically available fromcommercial services, the American Tissue Culture Collection or frompublished literature.

In one embodiment, the host used for expression of aninterferon-associated antigen binding protein is a non-human transgenicanimal or transgenic plant.

Interferon-associated antigen binding proteins of the invention can alsobe produced transgenically through the generation of a non-human animal(e.g., mammal) or plant that is transgenic for the sequences of interestand production of the interferon-associated antigen binding protein in arecoverable form therefrom. In connection with the transgenic productionin mammals, interferon-associated antigen binding proteins can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos 5,827,690, 5,756,687, 5,750,172, and5,741,957. Exemplary plant hosts are Nicotiana, Arabidopsis, duckweed,corn, wheat, potato, etc. Methods for expressing antibodies in plants,including a description of promoters and vectors, as well astransformation of plants is known in the art. See, e.g., U.S. Pat.6,517,529, herein incorporated by reference. In some embodiments,non-human transgenic animals or plants are produced by introducing oneor more nucleic acid molecules encoding an interferon-associated antigenbinding protein of the invention into the animal or plant by standardtransgenic techniques. See Hogan and U.S. Pat. 6,417,429. The transgeniccells used for making the transgenic animal can be embryonic stem cellsor somatic cells. The transgenic non-human organisms can be chimeric,nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hoganet al., Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., ColdSpring Harbor Press (1999); Jackson et al., Mouse Genetics andTransgenics: A Practical Approach, Oxford University Press (2000); andPinkert, Transgenic Animal Technology: A Laboratory Handbook, AcademicPress (1999). In some embodiments, the transgenic non-human animals havea targeted disruption and replacement by a targeting construct thatencodes the sequence(s) of interest. The interferon-associated antigenbinding proteins may be made in any transgenic animal. In a preferredembodiment, the non-human animals are mice, rats, sheep, pigs, goats,cattle or horses. The non-human transgenic animal expresses saidinterferon-associated antigen binding proteins in blood, milk, urine,saliva, tears, mucus and other bodily fluids.

In vitro production allows scale-up to give large amounts of the desiredinterferon-associated antigen binding proteins. Techniques for mammaliancell cultivation under tissue culture conditions are known in the artand include homogeneous suspension culture, e.g., in an airlift reactoror in a continuous stirrer reactor, or immobilized or entrapped cellculture, e.g., in hollow fibers, microcapsules, on agarose microbeads orceramic cartridges. If necessary and/or desired, a solution of aninterferon-associated antigen binding protein, can be purified by thecustomary chromatography methods, for example gel filtration,ion-exchange chromatography, chromatography over DEAE-cellulose and/or(immuno-) affinity chromatography.

One or more genes encoding an interferon-associated antigen bindingprotein can also be expressed in non-mammalian cells such as bacteria oryeast or plant cells. In this regard it will be appreciated that variousunicellular non-mammalian microorganisms such as bacteria can also betransformed; i.e. those capable of being grown in cultures orfermentation. Bacteria, which are susceptible to transformation, includemembers of the enterobacteriaceae, such as strains of Escherichia colior Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus;Streptococcus, and Haemophilus influenzae. It will further beappreciated that, when expressed in bacteria, interferon-associatedantigen binding proteins according to the invention or componentsthereof (i.e., agonistic anti-CD40 antibodies or agonistic antigenbinding fragments thereof, and IFNs or functional fragments of IFNs) canbecome part of inclusion bodies. The desired interferon-associatedantigen binding proteins may then need to be isolated, optionally alsorefolded, and purified.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker’s yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available. For expression in Saccharomyces, the plasmidYRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsmanet al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) iscommonly used. This plasmid already contains the TRP1 gene, whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)). The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

Therapeutic Vectors

A nucleic acid sequence encoding an interferon-associated antigenbinding protein can be inserted into a vector and used as a therapeuticvector, e.g., a vector that expresses an interferon-associated antigenbinding protein of the invention. The construction of suitable,functional expression constructs and therapeutic expression vectors isknown to one of ordinary skill in the art. Thus, in certain embodiments,the interferon-associated antigen binding protein may be administered toa subject by means of genetic delivery with RNA or DNA sequences, avector or vector system encoding the interferon-associated antigenbinding protein.

Therapeutic vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see, e.g., Chen et al., PNAS91:3054-3057 (1994)). The pharmaceutical preparation of a therapeuticvector can include the vector in an acceptable diluent.

An interferon-associated antigen binding protein encoding nucleic acid,or nucleic acids, can be incorporated into a gene construct to be usedas a part of a therapy protocol to deliver nucleic acids encoding aninterferon-associated antigen binding protein. Expression vectors for invivo transfection and expression of an interferon-associated antigenbinding protein are provided.

Expression constructs of such components may be administered in anybiologically effective carrier, e.g., any formulation or compositioncapable of effectively delivering the component nucleic acid sequence tocells in vivo, as are known to one of ordinary skill in the art.Approaches include, but are not limited to, insertion of the subjectnucleic acid sequence(s) in viral vectors including, but not limited to,recombinant retroviruses, adenovirus, adeno-associated virus and herpessimplex virus-1, recombinant bacterial or eukaryotic plasmids and thelike.

Retrovirus vectors and adeno-associated viral vectors can be used as arecombinant delivery system for the transfer of exogenous nucleic acidsequences in vivo, particularly into humans. Such vectors provideefficient delivery of genes into cells, and the transferred nucleicacids can be stably integrated into the chromosomal DNA of the host.

The development of specialized cell lines (termed “packaging cells”)which produce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see, e.g., Miller, Blood 76:271-78 (1990)). Areplication-defective retrovirus can be packaged into virions, which canbe used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, et al., (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14, and otherstandard laboratory manuals. Non-limiting examples of suitableretroviruses include pLJ, pZIP, pWE and pEM, which are known to those ofordinary skill in the art. Examples of suitable packaging virus linesinclude *Crip, *Cre, *2 and *Am. (See, for example, Eglitis, et al.,Science 230:1395-1398 (1985); Danos and Mulligan, Proc. Natl. Acad. Sci.USA 85:6460-6464 (1988); Wilson, et al., Proc. Natl. Acad. Sci. USA85:3014-3018 (1988); Armentano, et al., Proc. Natl. Acad. Sci. USA87:6141-6145 (1990); Huber, et al., Proc. Natl. Acad. Sci. USA88:8039-8043 (1991); Ferry, et al., Proc. Natl. Acad. Sci. USA88:8377-8381 (1991); Chowdhury, et al., Science 254:1802-1805 (1991);van Beusechem, et al., Proc. Natl. Acad. Sci. USA 89:7640-7644 (1992);Kay, et al., Human Gene Therapy 3:641-647 (1992); Dai, et al., Proc.Natl. Acad. Sci. USA 89:10892-10895 (1992); Hwu, et al., J. Immunol.150:4104-4115 (1993); U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286;PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

In another embodiment, adenovirus-derived delivery vectors are provided.The genome of an adenovirus can be manipulated such that it encodes andexpresses a gene product of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See, forexample, Berkner, et al., BioTechniques 6:616 (1988); Rosenfeld, et al.,Science 252:431-434 (1991); and Rosenfeld, et al., Cell 68:143-155(1992). Suitable adenoviral vectors derived from the adenovirus strainAd type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7etc.) are known to those of ordinary skill in the art. Recombinantadenoviruses can be advantageous in certain circumstances in that theyare not capable of infecting non-dividing cells and can be used toinfect a wide variety of cell types, including epithelial cells(Rosenfeld, et al. (1992), supra). Furthermore, the virus particle isrelatively stable and amenable to purification and concentration and, asabove, can be modified so as to affect the spectrum of infectivity.Additionally, introduced adenoviral DNA (and foreign DNA containedtherein) is not integrated into the genome of a host cell, but remainsepisomal, thereby avoiding potential problems that can occur as a resultof insertional mutagenesis in situ where introduced DNA becomesintegrated into the host genome (e.g., retroviral DNA). Moreover, thecarrying capacity of the adenoviral genome for foreign DNA is large (upto 8 kilobases) relative to other delivery vectors (Berkner, et al.(1998), supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).

Yet another viral vector system useful for delivery of a nucleic acidsequence encoding an interferon-associated antigen binding protein, isthe adeno-associated virus (AAV). AAV is a naturally occurring defectivevirus that requires another virus, such as an adenovirus or a herpesvirus, as a helper virus for efficient replication and a productive lifecycle. (For a review see Muzyczka, et al., Curr. Topics in Micro. andImmunol. 158:97-129 (1992)). It is also one of the few viruses that mayintegrate its DNA into non-dividing cells, and exhibits a high frequencyof stable integration (see for example Flotte, et al., Am. J. Respir.Cell. Mol. Biol. 7:349-356 (1992); Samulski, et al., J. Virol.63:3822-3828 (1989); and McLaughlin, et al., J. Virol. 62:1963-1973(1989)). Vectors containing as little as 300 base pairs of AAV can bepackaged and can integrate. Space for exogenous DNA is limited to about4.5 kb. An AAV vector such as that described in Tratschin, et al., Mol.Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells.A variety of nucleic acids have been introduced into different celltypes using AAV vectors (see for example Hermonat, et al., Proc. Natl.Acad. Sci. USA 81:6466-6470 (1984); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1985); Wondisford, et al., Mol. Endocrinol. 2:32-39 (1988);Tratschin, et al., J. Virol. 51:611-619 (1984); and Flotte, et al., J.Biol. Chem. 268:3781-3790 (1993)).

In addition to viral transfer methods, non-viral methods can also beemployed to cause expression of a nucleic acid sequence encoding aninterferon-associated antigen binding protein in the tissue of asubject. Most non-viral methods of gene transfer rely on normalmechanisms used by mammalian cells for the uptake and intracellulartransport of macromolecules. In some embodiments, non-viral deliverysystems rely on endocytic pathways for the uptake of the subject gene bythe targeted cell. Exemplary delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes. Other embodiments include plasmid injection systems such asare described in Meuli, et al., J. Invest. Dermatol. 116 (1):131-135(2001); Cohen, et al., Gene Ther 7 (22):1896-905 (2000); or Tam, et al.,Gene Ther. 7 (21):1867-74 (2000).

In clinical settings, the delivery systems can be introduced into asubject by any of a number of methods, each of which is familiar in theart. For instance, a pharmaceutical preparation of the delivery systemcan be introduced systemically, e.g., by intravenous injection. Specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by the delivery vehicle,cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the delivery vehicle can be introduced bycatheter (see, U.S. Pat. No. 5,328,470) or by stereotactic injection(e.g., Chen, et al., PNAS 91: 3054-3057 (1994)).

The pharmaceutical preparation of the therapeutic construct can consistessentially of the delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the delivery vehicle isimbedded. Alternatively, where the complete delivery system can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can comprise one or more cells, which producethe delivery system.

Methods of Treatment

In one aspect, the invention provides methods of treating a patient inneed thereof (e.g., a patient infected with HBV) comprisingadministering an effective amount of an interferon-associated antigenbinding protein, or a nucleic acid sequence (e.g., mRNA) that encodes aninterferon-associated antigen binding protein, as disclosed herein. Theinvention also provides for a use of an interferon-associated antigenbinding protein, or a nucleic acid sequence (e.g., mRNA) that encodes aninterferon-associated antigen binding protein, as disclosed herein, inthe preparation of a medicament. More specifically, the inventionprovides for a use of an interferon-associated antigen binding protein,or a nucleic acid sequence (e.g., mRNA) that encodes aninterferon-associated antigen binding protein, as disclosed herein, inthe preparation of a medicament for the treatment of disorders and/orsymptoms, e.g., HBV-related disorders and/or HBV-related symptoms. Incertain embodiments, the present invention provides kits and methods forthe treatment of disorders and/or symptoms, e.g., HBV-related disordersand/or HBV-related symptoms, in a mammalian subject in need of suchtreatment. In certain exemplary embodiments, the subject is a human.

The interferon-associated antigen binding proteins, or nucleic acidsequences that encode them, of the present invention are useful in anumber of different applications. For example, in one embodiment, thesubject interferon-associated antigen binding proteins, or nucleic acidsequences that encode them, are useful for reducing HBeAg release froman HBV-infected cell. In some embodiments, the interferon-associatedantigen binding proteins of the invention reduce HBeAg release byprimary hepatocytes in vitro by at least 10% at 1 ng/mL, at least 20% at1 ng/mL, at least 30 % at 1 ng/mL, at least 40% at 1 ng/mL, at least 50%at 1 ng/mL, at least 60 % at 1 ng/mL, at least 70% at 1 ng/mL, at least80% at 1 ng/mL, or at least 85% at 1 ng/mL. In some embodiments, theinterferon-associated antigen binding proteins of the invention reduceHBeAg release by primary hepatocytes in vitro by at least 12% at 1ng/mL. In some embodiments, the interferon-associated antigen bindingproteins of the invention reduce HBeAg release by primary hepatocytes invitro by up to 90% at 1 ng/mL. In related embodiments, theinterferon-associated antigen binding protein reduces HBeAg release withan EC₅₀ of less than 30 ng/mL, preferably with an EC₅₀ of less than 10ng/mL, more preferably with an EC₅₀ of less than 1 ng/mL.

In another embodiment, the subject interferon-associated antigen bindingproteins, or nucleic acid sequences that encode them, are useful forreducing pgRNA transcription of cccDNA in an HBV-infected cell.

In another embodiment, the subject interferon-associated antigen bindingproteins, or nucleic acid sequences that encode them, are useful forreducing one or more symptoms and/or complications associated with HBVinfection, as described herein (infra).

In certain embodiments, the subject interferon-associated antigenbinding proteins, or nucleic acid sequences that encode them, are usefulfor reducing one or more disorders, symptoms and/or complicationsassociated with chronic HBV infection, e.g., chronic inflammation of theliver (chronic hepatitis), leading to cirrhosis over a period of severalyears; hepatocellular carcinoma (HCC); development of membranousglomerulonephritis (MGN); risk of death; acute necrotizing vasculitis(polyarteritis nodosa), membranous glomerulonephritis, and papularacrodermatitis of childhood (Gianotti-Crosti syndrome); HBV-associatednephropathy (e.g., membranous glomerulonephritis); immune-mediatedhematological disorders (e.g., essential mixed cryoglobulinemia,aplastic anemia); and the like.

In certain embodiments, the subject interferon-associated antigenbinding proteins, or nucleic acid sequences that encode them, are usefulfor reducing one or more symptoms and/or complications associated withacute HBV infection, e.g., acute viral hepatitis (which begins withgeneral ill-health, loss of appetite, nausea, vomiting, body aches, mildfever, and dark urine, and then progresses to development of jaundice,fulminant hepatic failure, and/or serum-sickness-like syndrome); loss ofappetite; joint and muscle pain; low-grade fever; stomach pain; nausea;vomiting; jaundice; bloated stomach; and the like.

Accordingly, this invention also relates to a method of treating one ormore disorders, symptoms and/or complications associated with HBVinfection in a human or other animal by administering to such human oranimal an effective, non-toxic amount of an interferon-associatedantigen binding protein, or a nucleic acid sequence that encodes it. Oneskilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of aninterferon-associated antigen binding protein, or a nucleic acidsequence that encodes it, would be for the purpose of treating HBVinfection.

For example, a “therapeutically active amount” of aninterferon-associated antigen binding protein of the present inventionmay vary according to factors such as the disease stage (e.g., acute vs.chronic), age, sex, medical complications (e.g., HIV co-infection,immunosuppressed conditions or diseases) and weight of the subject, andthe ability of the interferon-associated antigen binding protein toelicit a desired response in the subject. The dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily, or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

In general, the compositions provided in the current invention may beused to prophylactically treat non-infected cells or therapeuticallytreat any HBV-infected cells comprising an antigenic marker that allowsfor the targeting of the HBV-infected cells by an interferon-associatedantigen binding protein.

Pharmaceutical Compositions and Administration Thereof

In certain embodiments, the interferon-associated antigen bindingproteins of the invention or nucleic acid sequences (including vectorsor vector systems) that encode them are comprised in a pharmaceuticalcomposition. Methods of preparing and administeringinterferon-associated antigen binding proteins, or nucleic acidsequences that encode them, of the current invention to a subject arewell known to or can be readily determined by those skilled in the artusing this specification and the knowledge in the art as a guide. Theroute of administration of the interferon-associated antigen bindingproteins, or nucleic acid sequences that encode them, of the currentinvention may be oral, parenteral, by inhalation or topical. The term“parenteral”, as used herein, includes intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, rectal or vaginaladministration. While all these forms of administration are clearlycontemplated as being within the scope of the current invention, a formfor administration would be a solution for injection, in particular forintravenous or intraarterial injection or drip. Usually, a suitablepharmaceutical composition for injection may comprise a buffering agent(e.g. acetate, phosphate or citrate buffer), a surfactant (e.g.polysorbate), optionally a stabilizing agent (e.g. human albumin), etc.In some embodiments, the buffering agent is acetate. In anotherembodiment, the buffering agent is formate. In yet another embodiment,the buffering agent is citrate. In related embodiments, the surfactantmay be selected from the list comprising pluronics, PEG, sorbitanesters, polysorbates, triton, tromethamine, lecithin, cholesterol andtyloxapal. In preferred embodiments, the surfactant is polysorbate. Inmore preferred embodiments, the surfactant is polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80 or polysorbate 100,preferably polysorbate 20 or polysorbate 80.

In some embodiments, the interferon-associated antigen binding proteins,or nucleic acid sequences that encode them, can be delivered directly tothe site of the adverse cellular population (e.g., the liver) therebyincreasing the exposure of the diseased tissue to the therapeutic agent.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the compositions and methods of the current invention,pharmaceutically acceptable carriers include, but are not limited to,0.01-0.1 M, e.g., 0.05 M phosphate buffer, or 0.8% saline. Other commonparenteral vehicles include sodium phosphate solutions, Ringer’sdextrose, dextrose and sodium chloride, lactated Ringer’s, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer’s dextrose, andthe like. Preservatives and other additives may also be present such asfor example, antimicrobials, antioxidants, chelating agents, and inertgases and the like. More particularly, pharmaceutical compositionssuitable for injectable use include sterile aqueous solutions (wherewater-soluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In suchcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and will typically be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, isotonic agents will be included, for example, sugars,polyalcohols, such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound such as an interferon-associatedantigen binding protein, or a nucleic acid sequence encoding saidinterferon-associated antigen binding protein, of the present inventionby itself or in combination with other active agents in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle, which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, which yields a powder of an activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparations for injections areprocessed, filled into containers such as ampules, bags, bottles,syringes or vials, and sealed under aseptic conditions according tomethods known in the art. Further, the preparations may be packaged andsold in the form of a kit. Such articles of manufacture will typicallyhave labels or package inserts indicating that the associatedcompositions are useful for treating a subject suffering from HBVinfection.

Effective doses of the compositions of the present invention, for thetreatment of the above described HBV infection-related conditions varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Usually, the patientis a human, but non-human mammals including transgenic mammals, inparticular non-human primates, can also be treated. Treatment dosagesmay be titrated using routine methods known to those of skill in the artto optimize safety and efficacy.

For treatment with an interferon-associated antigen binding protein, thedosage can range, e.g., from about 0.0001 to about 100 mg/kg, and moreusually about 0.01 to about 5 mg/kg (e.g., about 0.02 mg/kg, about 0.25mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 2 mg/kg,etc.), of the host body weight. For example, dosages can be about 1mg/kg body weight or about 10 mg/kg body weight or within the range ofabout 1 to about 10 mg/kg, e.g., at least about 1 mg/kg. Dosesintermediate in the above ranges are also intended to be within thescope of the current invention. Subjects can be administered such dosesdaily, on alternative days, weekly or according to any other scheduledetermined by empirical analysis. An exemplary treatment entailsadministration in multiple dosages over a prolonged period, for example,of at least six months. Additional exemplary treatment regimens entailadministration about once per every two weeks or about once a month orabout once every 3 to 6 months. Exemplary dosage schedules include about1 to about 10 mg/kg or about 15 mg/kg on consecutive days, about 30mg/kg on alternate days or about 60 mg/kg weekly.

Interferon-associated antigen binding proteins, or nucleic acidsequences expressing any of these, can be administered on multipleoccasions. Intervals between single dosages can be weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of interferon-associated antigen binding proteins of componentsthereof in the patient. Alternatively, interferon-associated antigenbinding proteins, or nucleic acid sequences expressing any of these canbe administered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the interferon-associated antigen binding proteinsin the patient.

The term “half-life” or “t_(½)”, as referred to herein, relates to thestability and/or the rate of excretion of a compound, such as theinterferon-associated antigen binding proteins of the invention. Inpractice, the half-life of a compound is usually measured in the serumand denotes the time after administration that the serum concentrationis 50% of the serum concentration at the time of administration. Theinterferon-associated antigen binding proteins of the invention arecharacterized by a long serum half-life in mice. In some embodiments,the half-life of the interferon-associated antigen binding protein is atleast 50 h, at least 60 h, at least 70 h, at least 80 h, at least 90 hor at least 100 h. In some embodiments, the half-life of theinterferon-associated antigen binding protein is at least 100 h. Inpreferred embodiments, the half-life of the interferon-associatedantigen binding protein in mice ranges from 116 to 158 h.

The half-life of a protein is related to its clearance. The term“clearance” or “clearance rate”, as used herein, refers to the volume ofplasma cleared of the protein per unit time. Clearance of theinterferon-associated antigen binding proteins of the invention is low.In some embodiments, clearance of the interferon-associated antigenbinding protein is below 10 mL/h/kg, below 5 mL/h/kg, below 2.5 mL/h/kg,below 1 mL/h/kg, or below 0.5 mL/h/kg. In some embodiments, clearance ofthe interferon-associated antigen binding protein is below 5 mL/h/kg. Insome embodiments, clearance of the interferon-associated antigen bindingprotein is below 1 mL/h/kg. In some embodiments, clearance of theinterferon-associated antigen binding protein in mice ranges from 0.28to 0.49 mL/h/kg.

The terms “volume of distribution”, “V_(D)”, “Vss” or “apparent volumeof distribution” as used herein refer to the theoretical volume thatwould be necessary to contain the total amount of an administeredcompound such as the interferon-associated antigen binding protein ofthe invention at the same concentration that it is observed in the bloodplasma and relates to the distribution of said compound between plasmaand the rest of the body after oral or parenteral dosing. In certainembodiments, the volume of distribution Vss of the interferon-associatedantigen binding protein is below 500 mL/kg, below 400 mL/kg, below 300mL/kg, below 200 mL/kg, or below 100 mL/kg. In some embodiments, thevolume of distribution Vss of the interferon-associated antigen bindingprotein is below 100 mL/kg. In some embodiments, the volume ofdistribution Vss of the interferon-associated antigen binding protein inmice ranges from 50 to 98 mL/kg.

Another related pharmacokinetic parameter is the systemic exposure. Asused herein, the terms “systemic exposure”, “AUC” or “area under thecurve” refer to the integral of the concentration-time curve. Systemicexposure might be represented by plasma (serum or blood) concentrationsor the AUCs of parent compound and/or metabolite(s). Theinterferon-associated antigen binding proteins of the inventioncirculate in the blood with higher systemic exposure (AUC (0-inf)) thantheir parental antibody (CP870,893). In some embodiments, the systemicexposure of the interferon-associated antigen binding protein is atleast 600 µg*h/mL, at least 700 µg*h/mL, at least 800 µg*h/mL, at least900 µg*h/mL or at least 1000 µg*h/mL, preferably at least 1000 µg*h/mL.In some embodiments, the systemic exposure of the interferon-associatedantigen binding protein in mice ranges from 1033 µg*h/mL to 1793µg*h/mL.

As previously discussed, an interferon-associated antigen bindingprotein of the present invention may be administered in apharmaceutically effective amount for the in vivo treatment of mammaliandisorders. In this regard, it will be appreciated that as disclosed aninterferon-associated antigen binding protein, will be formulated tofacilitate administration and promote stability of the active agent.

A pharmaceutical composition in accordance with the present inventioncan comprise a pharmaceutically acceptable, non-toxic, sterile carriersuch as physiological saline, nontoxic buffers, preservatives and thelike. A pharmaceutically effective amount of an interferon-associatedantigen binding protein typically is an amount sufficient to mediate oneor more of: a reduction of HBeAg release from an HBV-infected cell; areduction of pgRNA transcription in an HBV-infected cell; and astimulation of the IFN signaling pathway in an infected cell. Of course,the pharmaceutical compositions of the present invention may beadministered in single or multiple doses to provide for apharmaceutically effective amount of the interferon-associated antigenbinding protein.

In keeping with the scope of the present invention,interferon-associated antigen binding proteins, or nucleic acidsequences expressing any of them, may be administered to a human orother animal in accordance with the aforementioned methods of treatmentin an amount sufficient to produce a therapeutic effect. Theinterferon-associated antigen binding proteins, or nucleic acidsequences expressing any of them, can be administered to such human orother animal in a conventional dosage form prepared by combining theinterferon-associated antigen binding proteins, or nucleic acidsequences expressing any of them, with a conventional pharmaceuticallyacceptable carrier or diluent according to known techniques. It will berecognized by one of skill in the art that the form and character of thepharmaceutically acceptable carrier or diluent is dictated by the amountof active ingredient with which it is to be combined, the route ofadministration and other well-known variables. Those skilled in the artwill further appreciate that a cocktail comprising one or more speciesof interferon-associated antigen binding proteins, or nucleic acidsequences expressing any of them, described in the current invention mayprove to be effective.

It is to be understood that the methods described in this invention arenot limited to particular methods and experimental conditions disclosedherein as such methods and conditions may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Furthermore, the experiments described herein, unless otherwiseindicated, use conventional molecular and cellular biological andimmunological techniques within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008),including all supplements, Molecular Cloning: A Laboratory Manual(Fourth Edition) by MR Green and J. Sambrook and Harlow et al.,Antibodies: A Laboratory Manual, Chapter 14, Cold Spring HarborLaboratory, Cold Spring Harbor (2013, 2nd edition).

Unless otherwise defined, scientific and technical terms used hereinhave the meanings that are commonly understood by those of ordinaryskill in the art. In the event of any latent ambiguity, definitionsprovided herein take precedent over any dictionary or extrinsicdefinition. Unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. The useof “or” means “and/or” unless stated otherwise. The use of the term“including”, as well as other forms, such as “includes” and “included,”is not limiting. The use of the term “comprising” shall include the term“consisting of” unless stated otherwise.

Generally, nomenclature used in connection with cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein is well-knownand commonly used in the art. The methods and techniques provided hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. Enzymatic reactions and purification techniques areperformed according to manufacturer’s specifications, as commonlyaccomplished in the art or as described herein. The nomenclatures usedin connection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention using this disclosure as a guide. Having now described certainembodiments in detail, the same will be more clearly understood byreference to the following examples, which are included for purposes ofillustration only and are not intended to be limiting.

EXAMPLES Example I Generation of Interferon-Fused Antibodies (IFA) andCharacterization on Reporter cells I.a - IFA Design

The sequence combinations of exemplary IFAs, designed with CP870,893agonistic anti-CD40 antibody as backbone antibody, with the location ofIFNs and the nature of the linkers are listed in Table 7 and Table 8.IFN was fused via a linker at the N- or the C-terminal part of the LightChain (LC) or the Heavy Chain (HC), as indicated in Table 7. Nucleicacids encoding the HC, the LC or the fusions were synthesized withoptimized mammalian expression codons and cloned into a eukaryoticexpression vector such as pcDNA3.1 (Invitrogen). FIG. 2A depicts anexemplary map of a pcDNA3.1 plasmid encoding Seq ID NO 32 under thecontrol of the pCMV promoter.

I.b - IFA Production

The Freestyle 293-F cells (Invitrogen) were transiently cotransfectedwith plasmids encoding both HC and LC at a HC/LC ratio of 4/6. Six daysafter transfection, the supernatant was collected, centrifuged andfiltered through 0.22 µm filters. Purification process was performed intwo purification steps, on AktaExpress chromatography system (GEHealthcare) using Protein A MabSelect Sure 5 mL 1.6/2.5 cm column (GEHealthcare) at a Flow rate of 5 mL/min. Sample binding was done inD-PBS1X pH 7.5 buffer, and elution with Glycine/HCl 0.1 M pH 3.0 buffer.Elution peak was stored in a loop then injected on HiTrap desalting26/10 column (GE Healthcare) with a flow rate of 10 mL/min in D-PBS1XpH7.5 buffer. Elution peak was collected on a 96-well microplate (2 mLfractions). Pool was performed according to the UV peak profile. Afterfiltration on 0.22 µm filters (Sartorius MiniSart), quality control wasperformed including Bacterial Endotoxins using Endosafe® nexgen-PTS™(Charles River), size exclusion Chromatography: using SEC 200 Increase10/300 column (GE Healthcare) to determine purity and oligomers andSDS-PAGE under reducing and non-reducing conditions on NuPAGE gel System(Invitrogen) in MES SDS running buffer. The production yield isindicated in Table 8. For some IFAs, the production yield was very low.In that case, the agonistic CD40 activity and the IFN activity wereassessed directly using the supernatant containing IFAs without anyfurther purification.

Reduced SDS-PAGE analysis of purified IFAs indicated the presence of twomajor bands corresponding to the HC and the LC. When the IFN (whateverthe IFN family member) was fused to the HC, a shift of its molecularweight was observed and the same phenomenon was observed for the LCsfused with any IFN (FIG. 2B).

I.c - IFA Characterization on Reporter Cells

HEK-Blue™ CD40L cells (InvivoGen Cat. #: hkb-cd40) or HEK-Blue™ IFN-α/βcells (InvivoGen, Cat. #: hkb-ifnαβ), were used to monitor,respectively, the activation of the NFκB pathway by CD40 agonists or ofthe IFN pathway induced by type I-IFN.

HEK-Blue™ CD40L cells were generated by stable transfection of HEK293cells with the human CD40 gene and a NFκB-inducible Secreted EmbryonicAlkaline Phosphatase (SEAP) construct (Invivogen) to measure thebioactivity of CD40 agonists. Stimulation of CD40 leads to NFκBinduction and then production of SEAP, which is detected in thesupernatant using QUANTI-Blue™ (Invivogen, Cat. # rep-qbs2).

HEK-Blue™ IFN-cells are designed to monitor the activation of theJAK/STAT/ISGF3 pathways induced by type I-IFNs. Activation of thispathway induces the production and release of SEAP. Levels of SEAP arereadily assessable in the supernatant using QUANTI-Blue™.

HEK-Blue™ IFN-α/β are used to monitor the activity of human IFNα orIFNβ.

Cells were seeded in 96-well plates (50,000 cells per well) andstimulated with the indicated concentration for each IFA or controls andincubated at 37° C. for 24 h. Supernatants were then collected andlevels of SEAP were quantified after incubation of the supernatant forabout 30 min with QuantiBlue™ and Optical Density (O.D.) assessment at620 nm on an Ensight plate reader or PheraStar (Lab Biotech).

HEK-Blue™ Dual IFN-y cells (InvivoGen, Cat. #: hkb-ifng) or HEK-Blue™IFN-λ, (InvivoGen, Cat. #: hkb-ifnl) may be used to respectively monitorthe activity of type II- and type III-IFNs. HEK-Blue™ IFN-λ, cells aredesigned to monitor the activity of IFNλ. HEK-Blue™ Dual IFN-y cellsallow the detection of bioactive human IFNγ.

I.d - Functional Activities of IFNα/β-based IFAs on Reporter Cells

FIG. 3 shows examples of dose responses of IFAs, where IFNβ or a mutatedversion thereof as specified in Table 7 was fused to the HC as indicatedin Table 7, on HEK-Blue™ CD40L (FIGS. 3A-3B) and HEK-Blue™ IFN-α/β cells(FIGS. 3C-3D). Agonistic anti-CD40 activities of IFAs are summarized inTable 8 and examples are shown in FIG. 3A and FIG. 3B. Results indicatethat all tested IFAs are functional to activate both the CD40 pathwayand the IFN-α/β pathway in a dose dependent manner. For fusions to theC-terminus of the HC or LC, the EC₅₀ values for agonistic CD40 areranging from 11.1 ng/mL to 192 ng/mL (Table 8). The mean EC₅₀ value forthe parental antibody is 48 ng/mL and 57 ng/mL in the experiment shownin FIG. 3 . IFAs with the IFN fused to the N-terminus of the HC or theLC were also able to activate the CD40 pathway, but the precise EC₅₀values could not be determined for these IFAs since the activity wasdirectly determined from the supernatant and not using purified proteins(FIG. 3B).

The IFN activity of various IFAs is summarized in Table 8 and examplesare shown in FIGS. 3C to 3D. For fusions of IFNβ or mutated IFNβ (asspecified in Table 7) to the C-terminus of the HC or LC, the IFNactivity is variable depending on the linker sequence with EC₅₀ valuesranging from 0.14 ng/mL to 4.5 ng/mL (FIG. 3C and Table 8). FIG. 3Dshows that IFAs with IFNβ fused to the N-terminal part exhibit high IFNactivity. The parental antibody used as negative control did not showany activity, whereas recombinant IFNβ did show a strong dose-dependentresponse. Altogether, these results demonstrate that fusion of IFNβ or amutated version thereof as specified in Table 7 to an antibody,regardless the location, maintain both biological functions, althoughwith differences in terms of potencies.

FIG. 4 shows examples of dose responses of IFAs, where IFNα was fused tothe HC or the LC as indicated in Table 7, on HEK-Blue™ CD40L (FIG. 4Aand FIG. 4C) and HEK-Blue™ IFN-α/β cells (FIG. 4B and FIG. 4D). Resultsindicate that all tested IFAs are functional to activate both the CD40pathway and the IPNα/β pathway in a dose-dependent manner. Surprisingly,for all the IFNα-based IFAs, the potency on CD40 pathway wasreproducibly higher than that of the parental antibody. The EC₅₀ valuesfor IFNα-based IFAs ranged from 11.1 ng/mL to 22.7 ng/mL and the EC₅₀for CP870,893 ranged from 30 ng/mL to 80 ng/mL (mean EC₅₀ value: 48ng/mL).

The IFN activity of IFAs is variable depending on the linker sequencewith EC₅₀ values ranging from 1.6 ng/mL to 5.1 ng/mL. In the same assay,PEGylated IFNα2a (Pegasys®) was also active in a dose-dependent mannerwith an EC₅₀ value of around 1 ng/mL.

I.e - Generation and Characterization of IFAs Without the Fc Region

Suitable constructs according to the invention can also beinterferon-associated antigen binding proteins without an Fc region. Aconstruct encoding the heavy chain of the fab fragment of CP870,893fused to a TEV-His tag was designed (SEQ ID NO 50) and cloned into theexpression plasmid pcDNA3.1. This construct is cotransfected in HEKcells as described earlier, with LCs fused via different linkers todifferent IFNs such as SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 34, SEQ IDNO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 41, SEQ ID NO 42, or SEQ IDNO 43. Proteins and/or supernatants are evaluated in reporter cellsand/or their effect on HBV infection in PHHs. It will be understood byone of skill in the art that constructs for use in therapy will nolonger contain the TEV-His tag. These constructs are likewiseembodiments of the invention. Interferon-associated antigen bindingproteins without the Fc part will be active against HBV infection. TwoIFAs were then produced and their functional characterization isdescribed in Example V: IFA50: (SEQ ID NO 41) + (SEQ ID NO 50) andIFA51: (SEQ ID NO 42) + (SEQ ID NO 50).

Example II Effect of IFAs on HBV Infected Primary Hepatocytes II. a -Effect of IFAs on HBV Infection in Primary Human Hepatocytes

The effect of IFAs on HBV infection in primary human hepatocytes (PHHs)was investigated. PHH cells were plated in 96-well plates (70,000cells/well) in William’s E GlutaMAX media (32551-020, Gibco)supplemented with 10% fetal calf serum (FCS) (SH30066.02, Hyclone),insulin (19278-5ML, Sigma), hydrocortisone (H2270-100MG, Sigma) andPenicillin/Streptomycin (15140, Gibco). Four hours later, cells wererinsed and media was replaced. The next day, media was replaced bymatrigel-containing media (0.25 mg/mL; 356231, Corning). Cells wereinfected 48 hours after plating with a MOI (Multiplicity Of Infection)of 500 to 1.000 vge/cell (viral genome equivalent) in InVitroGRO HImedium (Z99009, Bioreclamation IVT) supplemented with 5% FCS, 4% PEG8000 (81268, Sigma), 2% DMSO (DMSO-100ML, Sigma) and 1%Penicillin/Streptomycin. Sixteen hours post-infection, cells were washedthree times with PBS. Four days after infection, cells were keptuntreated or treated with serially diluted IFAs as indicated in thefigures. Three days after treatment, culture supernatants were collectedand kept at -80° C. for further protein detection.

II.b. - HBV E-antigen (HBeAg) Release Assessment

HBV e-antigen (HBeAg) levels in the cell culture supernatant weremeasured using ELISA as described by the manufacturer and resultsexpressed in PEI Units (HBeAg CLIA 96T/K: CL0312-2; Autobio) or inluminescence.

II.c. - HBV S-antigen (HBsAg) Release Assessment

Quantification of the HBsAg in the supernatant was carried out byfollowing the protocol of the AutoBio HBsAg CLIA kit (# CL0310-2), themain steps were: first the samples were diluted ⅕ in 1X PBS. Then 50 µLof standards, controls, and diluted samples were placed in the wells. 50µL of “Enzyme conjugate” solution were added to each well, followed byan incubation of one hour at 37° C. Subsequently, the plates were washed6 times with 300 µL of washing solution from the kit using the platewasher. Then 50 µL of “Substrate Solution” (volume-to-volume mix inreagents A and B) was added in each well and an incubation of 10 minutesin the dark was carried out. The plates were then read on a PHERASTARmicroplate reader (BMG Labtech) in Luminescence mode.

II.d. - pgRNA Quantification

The qPCR technique was used to compare the level of expression of pgRNAfrom infected cells treated with test compounds. pgRNA quantificationfrom infected cells was done in 96-well plates with the QuantStudio 12 KFlex. The cDNA was obtained by RT, followed by qPCR with TaqMan FastVirus assay in one step (ThermoFisher cat# 4444434). The results wereprocessed by the ΔΔCt method and normalized with the housekeeping geneGUSB in duplex. The pgRNA was amplified using the following primers andprobe: (forward: CCTCACCATACTGCACTCA, reverse: GAGGGAGTTCTTCTTCTAGG,AGTGTGGATTCGCACTCCTCCAGC as a probe). The GUSB gene was amplified usingthe TaqMan assay from Thermo Fisher (Hs99999908-ml).

II.e. - CXCL10 Release

CXCL10 release was assessed using an ELISA kit according to themanufacturer’s instruction (BioLegend 439904). Samples were diluted 1/50and luminescence was assessed on an EnSight microplate reader at 450 nm.

II.f - Effect of IFNα/β Based IFAs on HBV Infection

Several IFAs were tested for their abilities to reduce HBeAg secretionafter infection of PHH with HBV. In FIG. 5 , IFAs with IFNβ or a mutatedversion thereof fused at the C-terminus of the LC were used. Resultsindicate that all the tested IFAs strongly reduce HBeAg release. Indeed,even at the lowest concentration tested (1 ng/mL), depending on the IFA, 70% to 90% inhibition of HBeAg release was observed, demonstratingthat they are endowed with potent anti-viral effect. It is noteworthythat 100% inhibition could not be reached in this experiment, sincetreatment started four days after infection and at that time an existingpool of HBeAg (mRNA and protein) is already present in the cell andcontinue to be produced thereafter.

The effect of IFAs fused to IFNα were also tested in HBV-infected PHHs.FIG. 6 shows that these IFAs are very potent on HBV infection with EC₅₀values ranging from 0.06 ng/mL to 0.2 ng/mL for IFAs with IFNα2a fusedat the C-terminus of the HC (IFA25: 0.16 ng/mL, IFA26: 0.1 ng/mL; IFA27:0.06 ng/mL; and IFA38: ~0.2 ng/mL (~2.2 pM); FIG. 6A and FIG. 6C) andfrom 0.15 ng/mL to 0.36 ng/mL for IFAs with IFNα2a fused at theC-terminus of the LC (IFA28: 0.36 ng/mL; IFA29: 0.15 ng/mL; IFA30: 0.31ng/mL; and IFA39: ~0.3 ng/mL (~3 pM); FIG. 6B and FIG. 6C). To comparethe antiviral effect of Pegasys to IFA38 and IFA39, results areexpressed in pM and indicate that EC₅₀ for Pegasys is ~250 pM incomparison to ~2.2 pM for IFA38 and ~3 pM for IFA39, indicating thatIFAs are much more potent than Pegasys.

II.g - Short Terms Treatment Is Sufficient to Induce Potent Anti-viralActivity

To assess the effect of short term IFA treatment of primary hepatocytesinfected with HBV, cells were infected and incubated for 4 days, treatedwith IFA25, IFA27, IFA28, IFA30 or with Pegasys in a dose dependentmanner for 24h, washed and then incubated with fresh medium without anytreatment. After 3 days, supernatants were collected to assess the levelof HBeAG (FIG. 6E), HBsAG (FIG. 6F) and CXCL10 (FIG. 6H) release andcells were lysed and RNA extracted for the quantification of pgRNA (FIG.6G). Results indicate that all tested IFAs were able to inhibit HbeAGand HBsAG release as well as pgRNA expression in a dose dependentmanner. Pegasys alone was only able to inhibit HBeAG release and reducepgRNA levels. In this respect, IFAs are at least 2 logs more active thanPegasys on viral parameters. Surprisingly, although all tested IFAsshowed a dose dependent inhibition of HBsAg release, no reduction wasobserved with Pegasys even at the highest concentration. Analysis ofCXCL10, a biomarker of the IFN pathway, showed that IFAs are also muchmore potent than Pegasys.

Example III Cytokine Release III.a - Cytokine Release Assessment (CRA)From Human Whole Blood Cells

Whole blood cells (WBC) ex vivo stimulation assay was used toinvestigate release of cytokines following IFA stimulation. WBC werecollected from four healthy donors, diluted ⅓ in RPMI1640 (72400-021,Gibco) and distributed in sterile reaction tubes (300 µl). Cells wereleft unstimulated, stimulated with LPS (LipoPolySaccharide) K12(tlrl-eklps, Invivogen) at 10 ng/mL as a positive control or with IFAsat 1 µg/ml and incubated for 24 h at 37° C. Supernatants were thencollected and frozen at -20° C. until the day of analysis.

Human pro-inflammatory cytokines were analyzed using multiplexing MSDassay (K15067L-4) which measures Tumor Necrosis Factor (TNF)-α,Interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12/IL-23p40 and IFNγ.MSD plates were analyzed on the 1300 MESO QuickPlex SQ120 apparatus(MSD).

FIG. 7 depicts exemplary results from an in vitro Cytokine ReleaseAssessment of Human WBC either non-stimulated, treated with LPS or withIFA1.

[0021] Further results from testing IFNβ- /mutated IFNβ- and IFNα- basedIFAs are summarized in Tables 9a and 9b. Results show that for alldonors, LPS induces very high level of the inflammatory cytokines(IL-1β, TNF-α, IL-6, IL-12p40 and IFNγ). It also induced IP10 (CXCL10)which is a biomarker of the IFN pathway and moderate level of IL-10. TwoIFNβ- (Table 9a) and six IFNα- (Table 9b) based IFAs were tested. All ofthem induced the biomarker IP10. However, they did not induce IL-10,IL-1β and IL-2, and they induced only very low to moderate level ofIFNy, IL-6 and TNF-α, thus suggesting a favorable safety profile withregard to the induction of inflammatory cytokines.

Example IV Pharmacokinetic Studies IV.a - ELISA Assay Development forIFA Quantifications

For the ELISA quantification 96-wells plates (PLATES 96 wells Maxisorp,THERMO Scientifique; 442404) were coated overnight at 4° C. with 100 µlof recombinant human CD40/TNFRSF5 Fc Chimera Protein, consisting of theextracellular domain of human CD40 fused to the Fc part of human IgG1(CD40-Fc; R&D Systems; 1493-CDB-050) at 0.5 µg/mL in Sodium Carbonate(0.05 M, pH 9.6, C-3041, Sigma). After emptying by flipping, plates werethen incubated for 1 hour at 37° C. with PBS - 0.05% Tween20 - 1% Milk(SIGMA; 70166-500 g) followed by washing with PBS-0.05% Tween20. Samplesand controls (100 µl of ½ serial dilutions) were then incubated for 90minutes at 37° C. followed by three washes (PBS - 0.05% Tween20) andincubation with a secondary anti-IgG2-conjugate HRP (1/5000, ab99779,Abcam) antibody or anti-IFNα conjugate HRP (1/1000, eBIOSCIENCE/Invitrogen; BMS216MST) in PBS - 0.05% Tween20 - 1% Milk. After threewashes with PBS, 0.05% Tween2, TMB (Tetramethylbenzidin, TebuBio;TMBW-1000-01) was added and the plates incubated for 20 minutes in thedark. The reaction was stopped by adding 1 M HCl. Plates were read at450-650 nm with an Ensight plate reader (Perkin Elmer). Quantificationof Pegasys was assessed using similar protocol steps but using humanIFN-α matched antibody pairs from eBioscience/Invitrogen. Capture wasperformed using 100 µL of human anti-IFNα antibody(eBioscience/Invitrogen; BMS216MST), at 1 µg/mL in sodium carbonate(0.05 M,pH 9.6, C-3041, Sigma). For the detection, a secondary anti-IFNαconjugate HRP antibody (1/1000, Affymetrix eBioscience/BMS216MST;15501707) in PBS -0.05% Tween20 - 1% Milk was applied.

IV.b - in Vivo Bioavailability in Mice

To determine the PK parameters, CP870,893, IFA25, IFA26, IFA27, IFA28,IFA29 and IFA30 were administrated at 0.5 mg/kg and Pegasys at 0.3 mg/kgi.v. bolus to male CD1-Swiss mice and blood samples were collected atdifferent time points. Examples of quantification of circulatingmolecules using the ELISA approach described above and revealed withanti-IFNα-conjugated HRP are shown in FIGS. 8A and 8B, while examples ofquantification revealed with anti-IgG2-conjugated HRP are shown in FIG.8C; Pegasys quantification is shown in FIG. 8D. In one set ofexperiments summarized in Table 10A, PK parameters for CP870,893 wereexplored in a 7-day experiment and those for IFA27, IFA29 and IFA30 in10-day experiments (quantification for IFA27 was performed using 2different ELISA approaches). In another set of experiments summarized inTable 10B, the PK parameters for CP870,893 and IFA25, IFA26, IFA28 andPegasys were explored in 21-day experiments (quantification for IFA25was performed using 2 different ELISA approaches).

After a short distribution phase, the pharmacokinetic profiles of IFAsare characterized by a long serum half-life ranging from 116 to 218 h(Table 10A and Table 10B). Very similar PK profiles were obtained forthe 6 tested IFAs with high circulating level even ten days after singledose administration. The pharmacokinetic parameters summarized in Table10A/B indicate that these IFAs surprisingly circulate in the blood withhigher systemic exposure (AUC (0-inf)) ranging from 1033 µg.h/mL to 2552µg.h/mL for IFAs in comparison to 590 or 797 µg.h/mL, respectively, forthe parental antibody CP870,893 (up to 3.2 fold), also reflecting lowerclearance values for IFAs. The volume of distribution Vss was low andranked from 50 to 105 mL/kg, slightly higher than the plasma vascularvolume (50 mL/kg) in this species. For all IFAs, the clearance wasranked as low (0.28 to 0.49 mL/h/kg). Interestingly, the clearance ofPegasys (1.4 mL/hr/kg) is up to 7 fold higher than clearance of IFAs(e.g., 0.2 mL/hr/kg for IFA27) demonstrating a higher systemic exposureof IFAs.

Example V V.a - Functional Activities of IFAs Without Fc Region onReporter Cells and HBV Infection

To determine whether the Fc part of IFAs is needed for activity, fusionsof IFNα to the C-terminal part of the LC associated with a Fab fragmentof the HC were designed and produced. IFNα was linked to the LC partwith a (G4S)2 (IFA50) or (G4S)3 (IFA51) linker.

Evaluation on HEK-Blue™ CD40L cells demonstrated that such IFAs stillexhibit agonistic CD40 activity (FIG. 9A) and activate the CD40 pathwaywith an EC₅₀ value of about 128 ng/ml (IFA 50) and 123 ng/mL (IFA51),respectively.

Evaluation of the IFN activity on HEK-Blue™ IFN-α/β cells showed thatboth tested IFAs exhibit IFN activity (FIG. 9B). EC₅₀ values arereported in Table 8B and are about 1.36 ng/ml for IFA50 and 1.43 ng/mLfor IFA51.

Both IFAs were tested on HBV infection as described earlier and bothIFAs exhibit potent anti-viral activity with EC₅₀ values of about 4.1 pM(IFA50) and 2.7 pM (IFA51), respectively.

V.b - Functional Activities ofIFNe Based IFAs on Reporter Cells and onHBV Infection

Fusions of CP870,893 to a third type I interferon (IFN epsilon; IFN_(ε))have also been designed and produced. Such IFAs were tested on HEK-Blue™CD40L cells and it could be demonstrated that they maintain agonisticCD40 activity. Results for one such IFA (IFA49) are shown in FIG. 10A.Evaluation on HEK-Blue™ hIFN-α/β cells (which are in fact activated byany type I interferon) showed that IFA49 is also able to activate theIFN-I-pathways (FIG. 10B). EC₅₀ values are reported in Table 8B. Inaddition, IFA49 was also tested on HBV infection in primary hepatocytesand showed similar activity to Pegasys (FIG. 10C).

These results demonstrate that IFAs with IFN_(ε) maintain both IFN andagonistic CD40 activity (i.e., are bifunctional) and have antiviralactivity.

V.c - Functional Activities of IFNω Based IFAs on Reporter Cells and onHBV Infection

Fusions of CP870,893 to a fourth type I interferon (IFN omega; IFN_(ω))have also been designed and produced. Such IFAs were tested on HEK-Blue™CD40L cells and results demonstrated that they maintain agonistic CD40activity. Results for one such IFA (IFA46) are shown in FIG. 11A.Evaluation on HEK-Blue™ hIFN-α/β cells (which are in fact activated byany type I interferon) showed that IFA46 is also able to activate theIFN-I-pathways (FIG. 11B). EC₅₀ values are reported in Table 8B. Inaddition, IFA46 was also tested on HBV infection in primary hepatocytesand showed similar activity to Pegasys (FIG. 11C).

These results demonstrate that IFAs with IFNω maintain both IFN andagonistic CD40 activity (i.e., are bifunctional) and have antiviralactivity.

V.d - Functional Activities ofIFNy Based IFAs on Reporter Cells on HBVInfection

Fusions of CP870,893 to type II Interferon (IFN gamma; IFNγ) have alsobeen designed and produced. Evaluation of these IFAs on HEK-Blue™ CD40Lcells demonstrate that they maintain agonistic CD40 activity, regardlessof whether IFNγ is linked to the C-terminal part of the LC (IFA42) or ofthe HC (IFA43) (FIG. 12A). Evaluation of these IFAs on HEK-Blue™-IFNγcells (FIG. 12B) showed that they are also able to activate theIFNγ-pathway. IFNγ activity differed somewhat between IFA42 (EC₅₀: 15ng/ml) and IFA43 (EC₅₀: < 0.01 ng/ml). EC₅₀ values are reported in Table8B. In addition, IFA42 and IFA43 were tested in a dose dependent manneron HBV infection in primary hepatocytes as described earlier. Resultsindicate that both IFAs reduce HbeAg release in a dose dependent manner(FIG. 12C), indicating that IFAs with type II-IFN are active on HBVinfection.

Taken together, these results demonstrate that IFAs with IFNγ maintainboth IFN and agonistic CD40 activity (i.e., are bifunctional) and haveanti-viral activity.

V.e - Functional Activities of IFNλ Based IFAs on Reporter Cells and onHBV Infection

Fusions of CP870,893 to type III Interferon (IFN lambda; IFNλ) have alsobeen designed and produced. These IFAs were tested on HEK-Blue™ CD40Lcells and results demonstrated that they also maintain agonistic CD40activity, regardless of whether IFNλ is linked to the C-terminal part ofthe LC (IFA44) or of the HC (IFA45) (FIG. 13A). Evaluation of these IFAson HEK-Blue™-IFNλ cells showed that they are also able to activate theIFNλ-pathway (FIG. 13B). EC₅₀ values are reported in Table 8B. Theseresults also demonstrate that IFAs with IFNλ maintain both IFN andagonistic CD40 activity (i.e., are bifunctional).

IFA44 and IFA45 were tested in a single dose in comparison to Pegasys onHBV infection in primary hepatocytes as described earlier. Resultsindicate that both types of IFAs reduce HbeAg release by 65% and 78%,respectively. Under these condition Pegasys inhibited HbeAg release by81%. These results indicate that IFAs with type III IFN are active onHBV infection with EC₅₀ values for both tested IFAs < 10 nM (FIG. 13C).

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention. Scope of the invention is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

Items

In view of the above, it will be appreciated that the present inventionalso relates to the following items:

1. An interferon-associated antigen binding protein comprising

-   (I) an agonistic anti-CD40 antibody or an agonistic antigen binding    fragment thereof, and-   (II) an Interferon (IFN) or a functional fragment thereof, wherein    the agonistic anti-CD40 antibody, or the agonistic antigen binding    fragment thereof, comprises    -   (a) three light chain complementarity determining regions (CDRs)        that are at least 90% identical to the CDRL1, CDRL2 and CDRL3        sequences within SEQ ID NO 3; and three heavy chain CDRs that        are at least 90% identical to the CDRH1, CDRH2 and CDRH3        sequences within SEQ ID NO 6; wherein each CDR is defined in        accordance with the Kabat definition, the Chothia definition,        the AbM definition, or the contact definition of CDR; preferably        wherein each CDR is defined in accordance with the CDR        definition of Kabat or the CDR definition of Chothia;    -   (b) three light chain complementarity determining regions (CDRs)        that are identical to the CDRL1, CDRL2 and CDRL3 sequences        within SEQ ID NO 3; and three heavy chain CDRs that are        identical to the CDRH1, CDRH2 and CDRH3 sequences within SEQ ID        NO 6; wherein each CDR is defined in accordance with the Kabat        definition, the Chothia definition, the AbM definition, or the        contact definition of CDR; preferably wherein each CDR is        defined in accordance with the CDR definition of Kabat or the        CDR definition of Chothia;    -   (c) a heavy chain or a fragment thereof comprising a        complementarity determining region (CDR) CDRH1 that is at least        90% identical to SEQ ID NO 56, a CDRH2 that is at least 90%        identical to SEQ ID NO 57, and a CDRH3 that is at least 90%        identical to SEQ ID NO 58; and a light chain or a fragment        thereof comprising a CDRL1 that is at least 90% identical to SEQ        ID NO 52, a CDRL2 that is at least 90% identical to SEQ ID NO        53, and a CDRL3 that is at least 90% identical to SEQ ID NO 54;    -   (d) a heavy chain or a fragment thereof comprising a        complementarity determining region (CDR) CDRH1 that is identical        to SEQ ID NO 56, a CDRH2 that is identical to SEQ ID NO 57, and        a CDRH3 that is identical to SEQ ID NO 58; and        -   a light chain or a fragment thereof comprising a CDRL1 that            is identical to SEQ ID NO 52, a CDRL2 that is identical to            SEQ ID NO 53, and a CDRL3 that is identical to SEQ ID NO 54;    -   (e) a light chain variable region V_(L) comprising the sequence        as set forth in SEQ ID NO 51, or a sequence at least 90%        identical thereto; and/or a heavy chain variable region V_(H)        comprising the sequence as set forth in SEQ ID NO 55, or a        sequence at least 90% identical thereto;    -   (f) a Fab region heavy chain comprising an amino acid sequence        as set forth in SEQ ID NO 12, or a sequence at least 90%        identical thereto; or    -   (g) a light chain (LC) that comprises a sequence as set forth in        SEQ ID NO 3, or a sequence at least 90% identical thereto;        and/or a heavy chain (HC) that comprises a sequence selected        from the group consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO        49 and SEQ ID NO 48, or a sequence at least 90% identical        thereto.

2. The interferon-associated antigen binding protein according to item1, wherein the HC comprises the sequence as set forth in SEQ ID NO 6, ora sequence at least 90% identical thereto.

3. The interferon-associated antigen binding protein according to item1, wherein the HC comprises the sequence as set forth in SEQ ID NO 9, ora sequence at least 90% identical thereto.

4. The interferon-associated antigen binding protein according to item1, wherein the HC comprises the sequence as set forth in SEQ ID NO 49,or a sequence at least 90% identical thereto.

5. The interferon-associated antigen binding protein according to item1, wherein the HC comprises the sequence as set forth in SEQ ID NO 48,or a sequence at least 90% identical thereto.

6. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the IFN or the functional fragmentthereof is a human interferon.

7. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the IFN or the functional fragmentthereof is selected from the group consisting of a Type I IFN, a Type IIIFN and a Type III IFN, or a functional fragment thereof.

8. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the IFN or the functional fragmentthereof is a Type I IFN, or a functional fragment thereof.

9. The interferon-associated antigen binding protein according to item8, wherein the type I IFN or the functional fragment thereof is IFNα,IFNβ, IFNω, or IFNε, or a functional fragment thereof.

10. The interferon-associated antigen binding protein according to item8, wherein the type I IFN or the functional fragment thereof is IFNα orIFNβ, or a functional fragment thereof.

11. The interferon-associated antigen binding protein according to item8, wherein the type I IFN or the functional fragment thereof is IFNω, ora functional fragment thereof.

12. The interferon-associated antigen binding protein according to item8, wherein the type I IFN or the functional fragment thereof is IFNε, ora functional fragment thereof.

13. The interferon-associated antigen binding protein according to anyone of the items 1 to 6, wherein the IFN or the functional fragmentthereof is IFNα, IFNβ, IFNγ, IFNλ, IFNω or IFNε, or a functionalfragment thereof.

14. The interferon-associated antigen binding protein according to item13, wherein the IFN or the functional fragment thereof is IFNα or IFNβ,or a functional fragment thereof.

15. The interferon-associated antigen binding protein according to item14, wherein the IFN or the functional fragment thereof is IFNα, or afunctional fragment thereof.

16. The interferon-associated antigen binding protein according to item15, wherein the IFN or functional fragment thereof is IFNα2a, or afunctional fragment thereof.

17. The interferon-associated antigen binding protein according to item16, wherein the IFNα2a comprises the sequence as set forth in SEQ ID NO17, or a sequence at least 90% identical thereto.

18. The interferon-associated antigen binding protein according to item14, wherein the IFN or the functional fragment thereof is IFNβ, or afunctional fragment thereof.

19. The interferon-associated antigen binding protein according to item18, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO14, or a sequence at least 90% identical thereto.

20. The interferon-associated antigen binding protein according to item18, wherein the IFNβ or the functional fragment thereof comprises one ortwo amino acid substitution(s) relative to SEQ ID NO 14, selected fromC17S and N80Q.

21. The interferon-associated antigen binding protein according to item20, wherein the IFNβ or the functional fragment thereof comprises theamino acid substitution C17S relative to SEQ ID NO 14.

22. The interferon-associated antigen binding protein according to item21, wherein the IFNβ comprises the amino acid sequence as set forth inSEQ ID NO 15.

23. The interferon-associated antigen binding protein according to item20, wherein the IFNβ or the functional fragment thereof comprises theamino acid substitutions C17S and N80Q relative to SEQ ID NO 14.

24. The interferon-associated antigen binding protein according to item23, wherein the IFNβ comprises the amino acid sequence as set forth inSEQ ID NO 16.

25. The interferon-associated antigen binding protein according to item13, wherein the IFN or a functional fragment thereof is IFNγ or IFNλ, ora functional fragment thereof.

26. The interferon-associated antigen binding protein according to item25, wherein the IFN or a functional fragment thereof is IFNγ, or afunctional fragment thereof.

27. The interferon-associated antigen binding protein according to item26, wherein the IFNγ comprises the sequence as set forth in SEQ ID NO19, or a sequence at least 90% identical thereto.

28. The interferon-associated antigen binding protein according to item25, wherein the IFN or a functional fragment thereof is IFNλ, or afunctional fragment thereof.

29. The interferon-associated antigen binding protein according to item28, wherein the IFNλ or the functional fragment thereof is IFNλ2, or afunctional fragment thereof.

30. The interferon-associated antigen binding protein according to item29, wherein the IFNλ2 comprises the sequence as set forth in SEQ ID NO18, or a sequence at least 90% identical thereto.

31. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the IFN or the functional fragmentthereof is non-covalently associated with the agonistic anti-CD40antibody or the agonistic antigen binding fragment thereof.

32. The interferon-associated antigen binding protein according to item31, wherein the IFN or the functional fragment thereof is non-covalentlyassociated with the agonistic anti-CD40 antibody or the agonisticantigen binding fragment thereof via ionic, Van-der-Waals, and/orhydrogen bond interactions.

33. The interferon-associated antigen binding protein according to anyone of items 1 to 30, wherein the IFN or the functional fragment thereofis covalently associated with the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof.

34. The interferon-associated antigen binding protein according to item33, wherein the IFN or the functional fragment thereof is fused to theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof.

35. The interferon-associated antigen binding protein according to item34, wherein the IFN or the functional fragment thereof is fused to alight chain of the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof.

36. The interferon-associated antigen binding protein according to item35, wherein the IFN or the functional fragment thereof is fused to theN-terminus of the light chain of the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof.

37. The interferon-associated antigen binding protein according to item35, wherein the IFN or the functional fragment thereof is fused to theC-terminus of the light chain of the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof.

38. The interferon-associated antigen binding protein according to item34, wherein the IFN or the functional fragment thereof is fused to aheavy chain of the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof.

39. The interferon-associated antigen binding protein according to item38, wherein the IFN or the functional fragment thereof is fused to theN-terminus of the heavy chain of the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof.

40. The interferon-associated antigen binding protein according to item38, wherein the IFN or the functional fragment thereof is fused to theC-terminus of the heavy chain of the agonistic anti-CD40 antibody or theagonistic antigen binding fragment thereof.

41. The interferon-associated antigen binding protein according to anyone of items 34 to 40, wherein the agonistic anti-CD40 antibody or anagonistic antigen binding fragment thereof, and the IFN or thefunctional fragment thereof are fused to each other via a linker.

42. The interferon-associated antigen binding protein according to item41, wherein the interferon-associated antigen binding protein comprisesno amino acids other than those forming (I) said agonistic anti-CD40antibody, or agonistic antigen binding fragment thereof, (II) said IFNor functional fragment thereof and (III) said linker.

43. The interferon-associated antigen binding protein according to anyone of items 1 to 41, wherein the interferon-associated antigen bindingprotein comprises no amino acids other than those forming (I) saidagonistic anti-CD40 antibody, or agonistic antigen binding fragmentthereof and (II) said IFN or functional fragment thereof.

44. The interferon-associated antigen binding protein according to anyone of items 41 to 42, wherein the linker is a peptide linker.

45. The interferon-associated antigen binding protein according to item44, wherein the linker comprises at least 1, at least 2, at least 3, atleast 4, or at least 5 amino acids.

46. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 4 amino acids.

47. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 11 amino acids.

48. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 12 amino acids.

49. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 13 amino acids.

50. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 15 amino acids.

51. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 20 amino acids.

52. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 21 amino acids.

53. The interferon-associated antigen binding protein according to item45, wherein the linker comprises at least 24 amino acids.

54. The interferon-associated antigen binding protein according to item44, wherein the linker comprises up to 10, up to 20, up to 30, up to 40,up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 aminoacids.

55. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 80 amino acids.

56. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 40 amino acids.

57. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 24 amino acids.

58. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 21 amino acids.

59. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 20 amino acids.

60. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 15 amino acids.

61. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 13 amino acids.

62. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 12 amino acids.

63. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 11 amino acids.

64. The interferon-associated antigen binding protein according to item54, wherein the linker comprises up to 4 amino acids.

65. The interferon-associated antigen binding protein according to anyone of items 44 to 64, wherein the linker is selected from the groupcomprising acidic, basic and neutral linkers.

66. The interferon-associated antigen binding protein according to item65, wherein the linker is an acidic linker.

67. The interferon-associated antigen binding protein according to item65 or 66, wherein the linker comprises a sequence as set forth in SEQ IDNO 22 or SEQ ID NO 23.

68. The interferon-associated antigen binding protein according to item65, wherein the linker is a basic linker.

69. The interferon-associated antigen binding protein according to item65, wherein the linker is a neutral linker.

70. The interferon-associated antigen binding protein according to item65 or 69, wherein the linker comprises a sequence as set forth in SEQ IDNO 20, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.

71. The interferon-associated antigen binding protein according to anyone of items 44 to 70, wherein the linker is selected from the groupcomprising rigid, flexible and helix-forming linkers.

72. The interferon-associated antigen binding protein according to item71, wherein the linker is a rigid linker.

73. The interferon-associated antigen binding protein according to item71 or 72, wherein the linker comprises a sequence as set forth in SEQ IDNO 20, SEQ ID NO 22 or SEQ ID NO 23.

74. The interferon-associated antigen binding protein according to item71, wherein the linker is a flexible linker.

75. The interferon-associated antigen binding protein according to item71 or 74, wherein the linker comprises a sequence as set forth in SEQ IDNO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.

76. The interferon-associated antigen binding protein according to item71, wherein the linker is a helix-forming linker.

77. The interferon-associated antigen binding protein according to item71 or 76, wherein the linker comprises a sequence as set forth in SEQ IDNO 22 or SEQ ID NO 23.

78. The interferon-associated antigen binding protein according to anyone of items 44 to 66, 68, 69, 71, 72, 74 or 76, wherein the linkercomprises the amino acids glycine and serine.

79. The interferon-associated antigen binding protein according to item78, wherein the linker comprises the sequence as set forth in SEQ ID NO21, SEQ ID NO 24, SEQ ID NO 25, or SEQ ID NO 26.

80. The interferon-associated antigen binding protein according to item78, wherein the linker further comprises the amino acid threonine.

81. The interferon-associated antigen binding protein according to item80, wherein the linker comprises the sequence as set forth in SEQ ID NO21.

82. The interferon-associated antigen binding protein according to item44, wherein the linker comprises a sequence selected from the sequencesas set forth in SEQ ID NOs 20 to 26.

83. The interferon-associated antigen binding protein according to item82, wherein the linker comprises a sequence selected from the sequencesas set forth in SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.

84. The interferon-associated antigen binding protein according to item83, wherein the linker comprises a sequence as set forth in SEQ ID NO24.

85. The interferon-associated antigen binding protein according to item83, wherein the linker comprises a sequence as set forth in SEQ ID NO25.

86. The interferon-associated antigen binding protein according to item83, wherein the linker comprises a sequence as set forth in SEQ ID NO26.

87. The interferon-associated antigen binding protein according to anyone of items 41, 42 or 44 to 86, wherein the IFN or a functionalfragment thereof is fused to the C-terminus of a heavy chain of theagonistic anti-CD40 antibody, or the agonistic antigen binding fragmentthereof, via the linker as set forth in Table 3, in particular Table 3Aor Table 3B, more particularly Table 3A.

88. The interferon-associated antigen binding protein according to item87, wherein the heavy chain of the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof, comprises a sequence as setforth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49, SEQ ID NO 48, or SEQ IDNO 12.

89. The interferon-associated antigen binding protein according to items87 or 88, wherein the IFNα2a comprises the sequence as set forth in SEQID NO 17.

90. The interferon-associated antigen binding protein according to items87 or 88, wherein the IFNβ comprises the sequence as set forth in SEQ IDNO 14, SEQ ID NO 15 or SEQ ID NO 16.

91. The interferon-associated antigen binding protein according to item90, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO14.

92. The interferon-associated antigen binding protein according to item90, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO15.

93. The interferon-associated antigen binding protein according to item90, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO16.

94. The interferon-associated antigen binding protein according to item87 or 88, wherein the IFNγ comprises the sequence as set forth in SEQ IDNO 19.

95. The interferon-associated antigen binding protein according to item87 or 88, wherein the IFNλ2 comprises the sequence as set forth in SEQID NO 18.

96. The interferon-associated antigen binding protein according to anyone of items 87 to 95, wherein the interferon-associated antigen bindingprotein further comprises a light chain of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof.

97. The interferon-associated antigen binding protein according to item96, wherein the light chain comprises a sequence as set forth in SEQ IDNO 3.

98. The interferon-associated antigen binding protein according to anyone of items 41, 42 or 44 to 86, wherein the IFN or a functionalfragment thereof is fused to the N-terminus of a heavy chain of theagonistic anti-CD40 antibody, or the agonistic antigen binding fragmentthereof, via the linker as set forth in Table 4, in particular Table 4Aor Table 4B, more particularly Table 4A.

99. The interferon-associated antigen binding protein according to item98, wherein the heavy chain of the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof, comprises a sequence as setforth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48, SEQ ID NO49 or SEQ ID NO 50, preferably a sequence as set forth in SEQ ID NO 6,SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48 or SEQ ID NO 49.

100. The interferon-associated antigen binding protein according toitems 98 or 99, wherein the IFNα2a comprises the sequence as set forthin SEQ ID NO 17.

101. The interferon-associated antigen binding protein according toitems 98 or 99, wherein the IFNβ comprises the sequence as set forth inSEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.

102. The interferon-associated antigen binding protein according to item101, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO14.

103. The interferon-associated antigen binding protein according to item101, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO15.

104. The interferon-associated antigen binding protein according to item101, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO16.

105. The interferon-associated antigen binding protein according toitems 98 or 99, wherein the IFNγ comprises the sequence as set forth inSEQ ID NO 19.

106. The interferon-associated antigen binding protein according toitems 98 or 99, wherein the IFNλ2 comprises the sequence as set forth inSEQ ID NO 18.

107. The interferon-associated antigen binding protein according to anyone of items 98 to 106, wherein the interferon-associated antigenbinding protein further comprises a light chain of an agonisticanti-CD40 antibody, or an agonistic antigen binding fragment thereof.

108. The interferon-associated antigen binding protein according to item107, wherein the light chain comprises a sequence as set forth in SEQ IDNO 3.

109. The interferon-associated antigen binding protein according to anyone of items 41, 42 or 44 to 86, wherein the IFN or a functionalfragment thereof is fused to the C-terminus of a light chain of theagonistic anti-CD40 antibody, or the agonistic antigen binding fragmentthereof, via the linker as set forth in Table 5, in particular Table 5Aor Table 5B, more particularly Table 5A.

110. The interferon-associated antigen binding protein according to item109, wherein the light chain of the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof, comprises a sequence as setforth in SEQ ID NO 3.

111. The interferon-associated antigen binding protein according toitems 109 or 110, wherein the IFNα2a comprises the sequence as set forthin SEQ ID NO 17.

112. The interferon-associated antigen binding protein according toitems 109 or 110, wherein the IFNβ comprises the sequence as set forthin SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.

113. The interferon-associated antigen binding protein according to item112, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO14.

114. The interferon-associated antigen binding protein according to item112, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO15.

115. The interferon-associated antigen binding protein according to item112, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO16.

116. The interferon-associated antigen binding protein according toitems 109 or 110, wherein the IFNγ comprises the sequence as set forthin SEQ ID NO 19.

117. The interferon-associated antigen binding protein according toitems 109 or 110, wherein the IFNλ2 comprises the sequence as set forthin SEQ ID NO 18.

118. The interferon-associated antigen binding protein according to anyone of items 109 to 117, wherein the interferon-associated antigenbinding protein further comprises a heavy chain of an agonisticanti-CD40 antibody, or an agonistic antigen binding fragment thereof.

119. The interferon-associated antigen binding protein according to item118, wherein the heavy chain of the agonistic anti-CD40 antibodycomprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO12, SEQ ID NO 48, SEQ ID NO 49 or SEQ ID NO 50, preferably a sequence asset forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48 or SEQID NO 49.

120. The interferon-associated antigen binding protein according to anyone of items 41, 42 or 44 to 86, wherein the IFN is fused to theN-terminus of a light chain of the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof, via the linker as set forthin Table 6, in particular Table 6A or Table 6B, more particularly Table6A.

121. The interferon-associated antigen binding protein according to item120, wherein the light chain of the agonistic anti-CD40 antibody, or theagonistic antigen binding fragment thereof, comprises a sequence as setforth in SEQ ID NO 3.

122. The interferon-associated antigen binding protein according toitems 120 or 121, wherein the IFNα2a comprises the sequence as set forthin SEQ ID NO 17.

123. The interferon-associated antigen binding protein according toitems 120 or 121, wherein the IFNβ comprises the sequence as set forthin SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.

124. The interferon-associated antigen binding protein according to item123, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO14.

125. The interferon-associated antigen binding protein according to item123, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO15.

126. The interferon-associated antigen binding protein according to item123, wherein the IFNβ comprises the sequence as set forth in SEQ ID NO16.

127. The interferon-associated antigen binding protein according toitems 120 or 121, wherein the IFNγ comprises the sequence as set forthin SEQ ID NO 19.

128. The interferon-associated antigen binding protein according toitems 120 or 121, wherein the IFNλ2 comprises the sequence as set forthin SEQ ID NO 18.

129. The interferon-associated antigen binding protein according to anyone of items 120 to 128, wherein the interferon-associated antigenbinding protein further comprises a heavy chain of an anti-CD40antibody, or an agonistic antigen binding fragment thereof.

130. The interferon-associated antigen binding protein according to item129, wherein the heavy chain of the agonistic anti-CD40 antibodycomprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO12, SEQ ID NO 48, SEQ ID NO 49 or SEQ ID NO 50, preferably a sequence asset forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48 or SEQID NO 49.

131. The interferon-associated antigen binding protein according to anyone of items 1 to 130, wherein the interferon-associated antigen bindingprotein comprises a sequence selected from SEQ ID NO 28, SEQ ID NO 29,SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34,SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39,SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44,SEQ ID NO 45, SEQ ID NO 46 and SEQ ID NO 47.

132. The interferon-associated antigen binding protein according to item131, wherein the interferon-associated antigen binding protein comprisesa sequence selected from SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQID NO 41, SEQ ID NO 42 or SEQ ID NO 43.

133. The interferon-associated antigen binding protein according toitems 131 or 132, wherein the interferon-associated antigen bindingprotein is an interferon-fused agonistic anti-CD40 antibody or aninterferon-fused agonistic antigen binding fragment thereof comprisingone of the sequence combinations disclosed in Table 8, in particularTable 8A or Table 8B, more particularly Table 8A.

134. The interferon-associated antigen binding protein according to item133, wherein the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic antigen binding fragment thereof comprising the sequences asset forth in SEQ ID NO 38 and SEQ ID NO 3.

135. The interferon-associated antigen binding protein according to item133, wherein the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic antigen binding fragment thereof comprising the sequences asset forth in SEQ ID NO 39 and SEQ ID NO 3.

136. The interferon-associated antigen binding protein according to item133, wherein the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic antigen binding fragment thereof comprising the sequences asset forth in SEQ ID NO 40 and SEQ ID NO 3.

137. The interferon-associated antigen binding protein according to item133, wherein the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic antigen binding fragment thereof comprising the sequences asset forth in SEQ ID NO 41 and SEQ ID NO 9.

138. The interferon-associated antigen binding protein according to item133, wherein the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic antigen binding fragment thereof comprising the sequences asset forth in SEQ ID NO 42 and SEQ ID NO 9.

139. The interferon-associated antigen binding protein according to item133, wherein the interferon-associated antigen binding protein is aninterferon-fused agonistic anti-CD40 antibody or an interferon-fusedagonistic antigen binding fragment thereof comprising the sequences asset forth in SEQ ID NO 43 and SEQ ID NO 9.

140. The interferon-associated antigen binding protein according to anyone of items 1 to 139, wherein the interferon-associated antigen bindingprotein activates both the CD40 and an IFN pathway.

141. The interferon-associated antigen binding protein according to item140, wherein CD40 activity is determined using a whole blood surfacemolecule upregulation assay or an in vitro reporter cell assay.

142. The interferon-associated antigen binding protein according to item141, wherein CD40 activity is determined using an in vitro reporter cellassay, optionally using HEK-Blue™ CD40L cells.

143. The interferon-associated antigen binding protein according to anyone of items 140 to 142, wherein the interferon-associated antigenbinding protein activates the CD40 pathway with an EC₅₀ of less than400, 300, 200, 150, 100, 70, 60, 50, 40, 30, 25, 20, or 15 ng/mL.

144. The interferon-associated antigen binding protein according to item143, wherein the interferon-associated antigen binding protein activatesthe CD40 pathway with an EC₅₀ ranging from 10 to 200 ng/mL.

145. The interferon-associated antigen binding protein according to item144, wherein the interferon-associated antigen binding protein activatesthe CD40 pathway with an EC₅₀ ranging from 10 to 50 ng/mL, preferably 10to 30 ng/mL.

146. The interferon-associated antigen binding protein according to anyone of items 140 to 145, wherein the interferon-associated antigenbinding protein activates the IFN pathway with an EC₅₀ of less than 100,60, 50, 40, 30, 20, 10, or 1 ng/mL.

147. The interferon-associated antigen binding protein according to anyone of items 140 to 146, wherein the interferon-associated antigenbinding protein activates the IFN pathway with an EC₅₀ of less than 11ng/mL, preferably less than 6 ng/mL.

148. The interferon-associated antigen binding protein according to anyone of items 140 to 147, wherein the IFN pathway is the IFNα, IFNβ,IFNε, IFNγ, IFNω or IFNλ pathway.

149. The interferon-associated antigen binding protein according to item148, wherein IFNβ activity is determined using an in vitro reporter cellassay, optionally using HEK-Blue™ IFN-α/β cells.

150. The interferon-associated antigen binding protein according to item148, wherein IFNα activity is determined using an in vitro reporter cellassay, optionally using HEK-Blue™ IFN-α/β cells.

151. The interferon-associated antigen binding protein according to item148, wherein IFNγ activity is determined using an in vitro reporter cellassay, optionally using HEK-Blue™ Dual IFN-y cells.

152. The interferon-associated antigen binding protein according to item148, wherein IFNλ activity is determined using an in vitro reporter cellassay, optionally using HEK-Blue™ IFN-λ cells.

153. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the interferon-associated antigenbinding protein reduces HBeAg release by primary hepatocytes in vitro byat least 12% at 1 ng/mL.

154. The interferon-associated antigen binding protein for the use ofitem 153, wherein the interferon-associated antigen binding proteinreduces HBeAg release by primary hepatocytes in vitro by up to 90% at 1ng/mL.

155. The interferon-associated antigen binding protein according to item153, wherein the interferon-associated antigen binding protein reducesHBeAg release with an EC₅₀ of less than 30 ng/mL.

156. The interferon-associated antigen binding protein according to item155, wherein the interferon-associated antigen binding protein reducesHBeAg release with an EC₅₀ of less than 10 ng/mL.

157. The interferon-associated antigen binding protein according to item156, wherein the interferon-associated antigen binding protein reducesHBeAg release with an EC₅₀ of less than 1 ng/mL.

158. The interferon-associated antigen binding protein according to item156, wherein the interferon-associated antigen binding protein reducesHBeAg release with an EC₅₀ of less than 0.1 ng/mL.

159. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the interferon-associated antigenbinding protein is capable of upregulating the expression level of oneor more IFN pathway biomarkers in an HBV-infected cell, preferably atleast 1.5-fold, more preferably at least 2-fold, most preferably atleast 3-fold.

160. The interferon-associated antigen binding protein according to item159, wherein the IFN pathway biomarker is a chemokine.

161. The interferon-associated antigen binding protein according to item160, wherein the IFN pathway biomarker is the interferon stimulated geneISG20.

162. The interferon-associated antigen binding protein according to item160, wherein the IFN pathway biomarker is a C-X-C chemokine, selectedfrom the group consisting of CXCL9, CXCL10 and CXCL11.

163. The interferon-associated antigen binding protein according to item162, wherein the IFN pathway biomarker is CXCL10.

164. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the interferon-associated antigenbinding protein does not significantly upregulate the expression levelof one or more of IL10, IL1β and IL2 in an HBV-infected cell.

165. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the systemic exposure of theinterferon-associated antigen binding protein is increased compared toantibody CP870,893, preferably by at least 10%, more preferably by atleast 15%, most preferably by at least 25%.

166. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the systemic exposure of theinterferon-associated antigen binding protein is at least 1000µg^(∗)h/mL.

167. The interferon-associated antigen binding protein according to item166, wherein the systemic exposure of the interferon-associated antigenbinding protein ranges from 1033 µg^(∗)h/mL to 1793 µg^(∗)h/mL.

168. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the half-life of theinterferon-associated antigen binding protein is at least 100 h.

169. The interferon-associated antigen binding protein according to item168, wherein the half-life of the interferon-associated antigen bindingprotein ranges from 116 to 158 h.

170. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the clearance rate of theinterferon-associated antigen binding protein is below 0.5 mL/h/kg.

171. The interferon-associated antigen binding protein according to item170, wherein the clearance of the interferon-associated antigen bindingprotein ranges from 0.28 to 0.49 mL/h/kg.

172. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the volume of distribution Vss ofthe interferon-associated antigen binding protein is below 100 mL/kg.

173. The interferon-associated antigen binding protein according to item172, wherein the volume of distribution Vss of the interferon-associatedantigen binding protein ranges from 50 to 98 mL/kg.

174. The interferon-associated antigen binding protein according to anyone of the preceding items, wherein the interferon-associated antigenbinding protein is suitable for administration to a subject in needthereof by means of genetic delivery with nucleic acid sequencesencoding the interferon-associated antigen binding protein, or a vectoror vector system encoding the interferon-associated antigen bindingprotein.

175. A nucleic acid encoding the interferon-associated antigen bindingprotein as recited in any one of the preceding items.

176. A nucleic acid encoding the IFN or the functional fragment thereofbeing fused to the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof as recited in any one of items 34 to 174.

177. The nucleic acid according to item 176 encoding the IFN or thefunctional fragment thereof being fused to the light chain of theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof, and further encoding a heavy chain of the agonistic anti-CD40antibody or the agonistic antigen binding fragment thereof.

178. The nucleic acid according to item 177, wherein the heavy chain ofthe agonistic anti-CD40 antibody or the agonistic antigen bindingfragment thereof comprises a sequence as set forth in SEQ ID NO 6, SEQID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO12, SEQ ID NO 13, SEQ ID NO 48, SEQ ID NO 49 or SEQ ID NO 50, or anucleic acid sequence at least 95% identical to a nucleic acid encodingany of these sequences.

179. The nucleic acid according to item 176 encoding the IFN or thefunctional fragment thereof being fused to the heavy chain of theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof, and further encoding a light chain of the agonistic anti-CD40antibody or the agonistic antigen binding fragment thereof.

180. The nucleic acid according to item 179, wherein the light chain ofthe agonistic anti-CD40 antibody or the agonistic antigen bindingfragment thereof comprises a sequence as set forth in SEQ ID NO 3, SEQID NO 4 or SEQ ID NO 5, or a nucleic acid sequence at least 95%identical to a nucleic acid encoding any of these sequences.

181. A nucleic acid encoding an IFN or a functional fragment thereoffused to an agonistic anti-CD40 antibody or an agonistic antigen bindingfragment thereof according to any of the sequences set forth in SEQ IDNOs 28 to 47, or a nucleic acid sequence at least 95% identical to anucleic acid encoding any of these sequences.

182. The nucleic acid according to items 175 to 181, wherein the nucleicacid further encodes a purification tag.

183. The nucleic acid according to item 182, wherein the purificationtag is a His-tag.

184. The nucleic acid according to items 182 or 183, wherein the nucleicacid further encodes a cleavage site to cleave off the purification tag.

185. The nucleic acid according to item 184, wherein the nucleic acidcomprises a sequence encoding the amino acid sequence as set forth inSEQ ID NO 27.

186. The nucleic acid according to items 175 to 185, wherein the nucleicacid further encodes a signal peptide.

187. The nucleic acid according to item 186, wherein the signal peptideis a secretory signal peptide.

188. The nucleic acid according to item 186 or 187, wherein the signalpeptide comprises the sequence as set forth in SEQ ID NO: 1 or SEQ IDNO: 2.

189. The nucleic acid according to item 188, wherein the signal peptidecomprises the sequence as set forth in SEQ ID NO: 1.

190. The nucleic acid according to item 188, wherein the signal peptidecomprises the sequence as set forth in SEQ ID NO: 2.

191. A vector or a vector system comprising the nucleic acid accordingto any one of items 175 to 190.

192. A vector system comprising

-   (I) a first vector comprising a nucleic acid encoding an IFN or a    functional fragment thereof fused to a light chain of the agonistic    anti-CD40 antibody or the agonistic antigen binding fragment thereof    of the interferon-associated antigen binding protein of any of items    1 to 174 and a second vector comprising a nucleic acid encoding a    heavy chain of the agonistic anti-CD40 antibody or the agonistic    antigen binding fragment thereof of the interferon-associated    antigen binding protein of any one of items 1 to 174; or-   (II) a first vector comprising a nucleic acid encoding an IFN or a    functional fragment thereof fused to a heavy chain of the agonistic    anti-CD40 antibody or the agonistic antigen binding fragment thereof    of the interferon-associated antigen binding protein of any of items    1 to 174 and a second vector comprising a nucleic acid encoding a    light chain of the agonistic anti-CD40 antibody or the agonistic    antigen binding fragment thereof of the interferon-associated    antigen binding protein of any one of items 1 to 174.

193. A vector system comprising

-   (I) a first vector according to item 191 for the expression of the    IFN or the functional fragment thereof fused to the light chain of    the agonistic anti-CD40 antibody or the agonistic antigen binding    fragment thereof and a second vector for expression of the heavy    chain of the agonistic anti-CD40 antibody or the agonistic antigen    binding fragment thereof; or-   (II) a first vector according to item 191 for the expression of the    IFN or the functional fragment thereof fused to the heavy chain of    the agonistic anti-CD40 antibody or the agonistic antigen binding    fragment thereof and a second vector for expression of the light    chain of the agonistic anti-CD40 antibody or the agonistic antigen    binding fragment thereof.

194. A composition comprising the interferon-associated antigen bindingprotein according to any one of items 1 to 174, the nucleic acidaccording to any one of items 175 to 190, the vector according to item191 or the vector system according to any one of items 191 to 193.

195. The composition according to item 194, wherein the composition is apharmaceutical composition.

196. The composition according to item 195, wherein the pharmaceuticalcomposition is suitable for oral, parenteral, or topical administrationor for administration by inhalation.

197. The composition according to item 196, wherein the pharmaceuticalcomposition is suitable for oral administration.

198. The composition according to item 196, wherein the pharmaceuticalcomposition is suitable for topical administration.

199. The composition according to item 196, wherein the pharmaceuticalcomposition is suitable for administration by inhalation.

200. The composition according to item 196, wherein the pharmaceuticalcomposition is suitable for parenteral administration.

201. The composition according to item 200, wherein the pharmaceuticalcomposition is suitable for intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, rectal or vaginal administration.

202. The composition according to item 201, wherein the pharmaceuticalcomposition is suitable for injection, preferably for intravenous orintraarterial injection or drip.

203. The composition according to any one of items 195 to 202 comprisingat least one buffering agent.

204. The composition according to item 203, wherein the buffering agentis acetate, formate or citrate.

205. The composition according to item 204, wherein the buffering agentis acetate.

206. The composition according to item 204, wherein the buffering agentis formate.

207. The composition according to item 204, wherein the buffering agentis citrate.

208. The composition according to any one of items 195 to 207, whereinthe pharmaceutical formulation comprises a surfactant.

209. The composition according to item 208, wherein the surfactant isselected from the list comprising pluronics, PEG, sorbitan esters,polysorbates, triton, tromethamine, lecithin, cholesterol and tyloxapal.

210. The composition according to item 209, wherein the surfactant ispolysorbate.

211. The composition according to item 210, wherein the surfactant ispolysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 orpolysorbate 100.

212. The composition according to item 211, wherein the surfactant ispolysorbate 20.

213. The composition according to item 211, wherein the surfactant ispolysorbate 80.

214. The composition according to any one of items 195 to 213 comprisinga stabilizing agent, optionally wherein the stabilizing agent isalbumin.

215. A host cell comprising the nucleic acid according to any one ofitems 175 to 190, the vector according to item 191 or the vector systemaccording to any one of items 191 to 193.

216. A method of making the interferon-associated antigen bindingprotein according to any one of items 1 to 174, comprising culturing thehost cell according to item 215 and recovering saidinterferon-associated antigen binding protein.

217. A non-human transgenic animal or transgenic plant comprising thenucleic acid according to any one of items 175 to 190, the vectoraccording to item 191 or the vector system according to any one of items191 to 193, wherein the non-human transgenic animal or transgenic plantexpresses said nucleic acid.

218. A method of making the interferon-associated antigen bindingprotein according to any one of items 1 to 174, comprising the step ofisolating the interferon-associated antigen binding protein from thenon-human transgenic animal or transgenic plant according to item 217.

219. The interferon-associated antigen binding protein according to anyone of items 1 to 174, the nucleic acid according to any one of items175 to 190, the vector according to item 191, the vector systemaccording to any one of items 191 to 193 or the composition according toany one of items 195 to 214 for use as a medicament.

220. The interferon-associated antigen binding protein according to anyone of items 1 to 174, the nucleic acid according to any one of items175 to 190, the vector according to item 191, the vector systemaccording to any one of items 191 to 193 or the composition according toany one of items 195 to 214 for use in treating hepatitis B virus (HBV)infection and/or for decreasing one or more symptoms of HBV infection ina patient.

221. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 220, wherein treating hepatitis B virus (HBV) infection comprisesdecreasing one or more symptoms of HBV infection in the patient.

222. The interferon-associated antigen binding protein according to anyone of items 1 to 174, the nucleic acid according to any one of items175 to 190, the vector according to item 191, the vector systemaccording to any one of items 191 to 193 or the composition according toany one of items 195 to 214 for use in reducing the HBV viral load in anHBV-infected cell culture or an HBV-infected patient compared to anuntreated HBV-infected cell culture or to the same patient beforetreatment.

223. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 222, wherein the HBV viral load is reduced by about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

224. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 222 or 223, wherein the HBV viral load is determined by PCR orqRT-PCR.

225. The interferon-associated antigen binding protein according to anyone of items 1 to 174, the nucleic acid according to any one of items175 to 190, the vector according to item 191, the vector systemaccording to any one of items 191 to 193 or the composition according toany one of items 195 to 214 for use in reducing the HBV viral titer inan HBV-infected cell culture or an HBV-infected patient compared to anuntreated HBV-infected cell culture or to the same patient beforetreatment.

226. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 225, wherein the HBV viral titer is reduced by about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

227. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 225 or 226, wherein the HBV viral titer is determined by PCR orqRT-PCR.

228. The interferon-associated antigen binding protein according to anyone of items 1 to 174, the nucleic acid according to any one of items175 to 190, the vector according to item 191, the vector systemaccording to any one of items 191 to 193 or the composition according toany one of items 195 to 214 for use in reducing transcription of HBVcccDNA in an HBV-infected cell culture or an HBV-infected patientcompared to an untreated HBV-infected cell culture or to the samepatient before treatment.

229. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 228, wherein transcription of cccDNA is reduced by about 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

230. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 228 or 229, wherein cccDNA transcription is determined by qPCR.

231. The interferon-associated antigen binding protein according to anyone of items 1 to 174, the nucleic acid according to any one of items175 to 190, the vector according to item 191, the vector systemaccording to any one of items 191 to 193 or the composition according toany one of items 195 to 214 for use in reducing the level of pre-genomicHBV RNA in an HBV-infected cell culture or an HBV-infected patientcompared to an untreated HBV-infected cell culture or to the samepatient before treatment.

232. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 231, wherein the level of pre-genomic HBV RNA is reduced by about20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%.

233. The interferon-associated antigen binding protein, the nucleicacid, the vector, the vector system or the composition for the use ofitem 231 or 232, wherein the level of pre-genomic HBV RNA is determinedby qRT-PCR.

1-18. (canceled)
 19. A composition comprising an interferon-associatedantigen binding protein comprising: (I) an agonistic anti-CD40 antibodyor an agonistic antigen binding fragment thereof, and (II) an Interferon(IFN) or a functional fragment thereof, wherein the IFN or functionalfragment thereof does not include Interferon-α2a (INF-α2a) or afunctional fragment thereof, wherein the agonistic anti-CD40 antibody,or the agonistic antigen binding fragment thereof, comprises a heavychain and a light chain, wherein the heavy chain comprises acomplementarity determining region 1 (CDRH1) comprising the amino acidsequence of SEQ ID NO: 56; a complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO: 57; and acomplementarity determining region 3 (CDRH3) comprising the amino acidsequence of SEQ ID NO: 58; and wherein the light chain comprises acomplementarity determining region 1 (CDRL1) comprising the amino acidsequence of SEQ ID NO: 52; a complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO: 53; and acomplementarity determining region 3 (CDRL3) comprising the amino acidsequence of SEQ ID NO:
 54. 20. The composition of claim 19, wherein theheavy chain comprises a heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 55 and the light chain comprises a lightchain variable domain comprising the amino acid sequence of SEQ ID NO:51.
 21. The composition of claim 19, wherein the heavy chain comprisesthe amino acid sequence of SEQ ID NO: 6 and the light chain comprisesthe amino acid sequence of SEQ ID NO:
 3. 22. The composition of claim19, wherein the agonistic anti-CD40 antibody, or the agonistic antigenbinding fragment thereof, comprises a Fab region heavy chain comprisingthe amino acid sequence of SEQ ID NO:
 12. 23. The composition of claim19, wherein the IFN or the functional fragment thereof is fused to thelight chain of the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof.
 24. The composition of claim 19, wherein theIFN or the functional fragment thereof is fused to a C-terminus of thelight chain of the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof.
 25. The composition of claim 19, wherein theIFN or the functional fragment thereof is fused to the heavy chain ofthe agonistic anti-CD40 antibody or the agonistic antigen bindingfragment thereof.
 26. The composition of claim 19, wherein the IFN orthe functional fragment thereof is fused to a C-terminus of the heavychain of the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof.
 27. The composition of claim 19, wherein theagonistic anti-CD40 antibody or the agonistic antigen binding fragmentthereof is fused to the IFN or the functional fragment thereof via alinker.
 28. The composition of claim 27, wherein the linker comprisesthe amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24,SEQ ID NO: 25 or SEQ ID NO:
 26. 29. The composition of claim 27, whereinthe linker comprises the amino acid sequence of SEQ ID NO: 24, SEQ IDNO: 25 or SEQ ID NO:
 26. 30. The composition of claim 19, wherein theIFN or the functional fragment thereof is selected from the groupconsisting of a Type I IFN, a Type II IFN and a Type III IFN, or afunctional fragment thereof.
 31. The composition of claim 30, whereinthe Type I IFN or the functional fragment thereof is IFNβ or afunctional fragment thereof.
 32. The composition of claim 31, whereinthe IFNβ comprises the amino acid sequence of SEQ ID NO: 14 or an aminoacid sequence having at least 90% identity to SEQ ID NO:
 14. 33. Thecomposition of claim 30, wherein the Type III IFN or the functionalfragment thereof is IFNλ or a functional fragment thereof.
 34. Thecomposition of claim 33, wherein the IFNλ or the functional fragmentthereof is IFNλ2 or a functional fragment thereof.
 35. The compositionof claim 34, wherein the IFNλ2 comprises the amino acid sequence of SEQID NO: 18 or an amino acid sequence having at least 90% identity to SEQID NO:
 18. 36. The composition of claim 19, wherein the composition is apharmaceutical composition.
 37. A method for treating hepatitis B virus(HBV) infection and/or reducing one or more symptoms of HBV infection ina subject comprising: administering an effective amount of thepharmaceutical composition of claim
 36. 38. The method of claim 37,wherein the one or more symptoms of HBV infection are associated withchronic inflammation of the liver, hepatocellular carcinoma, developmentof membranous glomerulonephritis, risk of death, acute necrotizingvasculitis, papular acrodermatitis of childhood, HBV-associatednephropathy, and/or immune-mediated hematological disorders.
 39. Themethod of claim 37, wherein the one or more symptoms of HBV infectionare associated with acute viral hepatitis.
 40. The method of claim 39,wherein the one or more symptoms of HBV infection associated with acuteviral hepatitis comprise a loss of appetite, nausea, vomiting, bodyaches, dark urine, jaundice, fulminant hepatic failure, low-grade fever,stomach pain, and/or bloated stomach.
 41. The method of claim 37,wherein the subject is a human.
 42. The method of claim 37, wherein theheavy chain variable domain comprises the amino acid sequence of SEQ IDNO: 55 and the light chain variable domain comprises the amino acidsequence of SEQ ID NO:
 51. 43. The method of claim 37, wherein the heavychain comprises the amino acid sequence of SEQ ID NO: 6 and the lightchain comprises the amino acid of SEQ ID NO:
 3. 44. The method of claim37, wherein the agonistic anti-CD40 antibody, or the agonistic antigenbinding fragment thereof, comprises a Fab region heavy chain comprisingthe amino acid sequence of SEQ ID NO:
 12. 45. The method of claim 37,wherein the agonistic anti-CD40 antibody or the agonistic antigenbinding fragment thereof is fused to the IFN or the functional fragmentthereof via a linker.
 46. The method of claim 45, wherein the linkercomprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 24, SEQ ID NO: 25, or SEQ ID NO:
 26. 47. The method of claim 37,wherein the IFN or the functional fragment thereof is selected from thegroup consisting of a Type I IFN, a Type II IFN, and a Type III IFN, ora functional fragment thereof.
 48. The method of claim 47, wherein theType I IFN or the functional fragment thereof is IFNβ or a functionalfragment thereof.
 49. The method of claim 48, wherein the IFNβ comprisesthe amino acid sequence of SEQ ID NO: 14 or an amino acid sequencehaving at least 90% identity to SEQ ID NO:
 14. 50. The method of claim47, wherein the Type III IFN or the functional fragment thereof is IFNλor a functional fragment thereof.
 51. The method of claim 50, whereinthe IFNλ or the functional fragment thereof is IFNλ2 or a functionalfragment thereof.
 52. The method of claim 51, wherein the IFNλ2comprises the amino acid sequence of SEQ ID NO: 18 or an amino acidsequence having at least 90% identity to SEQ ID NO:
 18. 53. A nucleicacid encoding an interferon-associated antigen binding protein, whereinthe interferon-associated antigen binding protein comprises: (I) anagonistic anti-CD40 antibody or an agonistic antigen binding fragmentthereof, and (II) an Interferon (IFN) or a functional fragment thereof,wherein the IFN or functional fragment thereof does not includeInterferon-α2a (IFN-α2a) or a functional fragment thereof, wherein theagonistic anti-CD40 antibody, or the agonistic antigen binding fragmentthereof, comprises a heavy chain and a light chain, wherein the heavychain comprises a complementarity determining region 1 (CDRH1)comprising the amino acid sequence of SEQ ID NO: 56; a complementaritydetermining region 2 (CDRH2) comprising the amino acid sequence of SEQID NO: 57; and a complementarity determining region 3 (CDRH3) comprisingthe amino acid sequence of SEQ ID NO: 58; and wherein the light chaincomprises a complementarity determining region 1 (CDRL1) comprising theamino acid sequence of SEQ ID NO: 52; a complementarity determiningregion 2 (CDRL2) comprising the amino acid sequence of SEQ ID NO: 53;and a complementarity determining region 3 (CDRL3) comprising the aminoacid sequence of SEQ ID NO:
 54. 54. A vector comprising the nucleic acidaccording to claim
 53. 55. A vector system comprising: (I) a firstvector comprising a nucleic acid encoding an Interferon (IFN), or afunctional fragment thereof, fused to a light chain of an agonisticanti-CD40 antibody, or an agonistic antigen binding fragment thereof,wherein the IFN or functional fragment thereof does not includeInterferon-α2a (IFN-a2a) or a functional fragment thereof, and whereinthe light chain of the agonistic anti-CD40 antibody or agonisticantigen-binding fragment thereof comprises a complementarity determiningregion 1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 52; acomplementarity determining region 2 (CDRL2) comprising the amino acidsequence of SEQ ID NO: 53; and a complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 54 and a secondvector comprising a nucleic acid encoding a heavy chain of an agonisticanti-CD40 antibody, or agonistic antigen binding fragment thereof,wherein the heavy chain of the agonistic anti-CD40 antibody or agonisticantigen-binding fragment thereof comprises a complementarity determiningregion 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 56; acomplementarity determining region 2 (CDRH2) comprising the amino acidsequence of SEQ ID NO: 57; and a complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO: 58; or (II) afirst vector comprising a nucleic acid encoding an IFN, or a functionalfragment thereof, fused to a heavy chain of an agonistic anti-CD40antibody, or an agonistic antigen binding fragment thereof, wherein theIFN or functional fragment thereof does not include Interferon-α2a(IFNα2a) or a functional fragment thereof, and wherein the heavy chainof the agonistic anti-CD40 antibody or agonistic antigen-bindingfragment thereof comprises a complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO: 56; acomplementarity determining region 2 (CDRH2) comprising the amino acidsequence of SEQ ID NO: 57; and a complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO: 58 and a secondvector comprising a nucleic acid encoding a light chain of an agonisticanti-CD40 antibody, or agonistic antigen binding fragment thereof,wherein the light chain of the agonistic anti-CD40 antibody or agonisticantigen-binding fragment thereof comprises a complementarity determiningregion 1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 52; acomplementarity determining region 2 (CDRL2) comprising the amino acidsequence of SEQ ID NO: 53; and a complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO:
 54. 56. Anisolated host cell comprising the nucleic acid according to claim 53.57. An isolated host cell comprising the vector according to claim 54.58. An isolated host cell comprising the vector system according toclaim 55.